Open Access

Characterization of exosomal release in bovine endometrial intercaruncular stromal cells

  • Yong Qin Koh1,
  • Hassendrini N. Peiris1,
  • Kanchan Vaswani1,
  • Sarah Reed1,
  • Gregory E. Rice1,
  • Carlos Salomon1 and
  • Murray D. Mitchell1Email author
Reproductive Biology and Endocrinology201614:78

https://doi.org/10.1186/s12958-016-0207-4

Received: 21 September 2016

Accepted: 25 October 2016

Published: 9 November 2016

Abstract

Background

Cell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation).

Methods

ICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry.

Results

Under hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2.

Conclusions

In summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.

Keywords

Bovine Intercaruncular Hypoxia Exosomes

Background

In dairy cattle, the average gestation length is approximately 282 days. The placenta is epitheliochorial, cotyledonary and non-deciduate [1]. Placentation is restricted to the aglandular maternal caruncles, where the fetal cotyledons come into contact with each other [2, 3]. They then form the placentome for maternal-fetal exchange of oxygen, nutrients and waste products. The glandular intercaruncular regions are associated with preserving the uterus in a state of quiescence and allowing a progressive uterine hypertrophy to accommodate the increasing needs of the growing feto-placental unit [4]. The uterine glands present in the intercaruncular endometrial areas secrete and release histotroph that is crucial for conceptus survival and growth [5] and is transported into the fetal circulation via the placental areolae. The establishment of a successful pregnancy requires the interactions between the endometrial cells and the early conceptus during maternal recognition of pregnancy [6, 7].

Cells located within intercaruncular region and associated with maternal fetal crosstalk include cells of stromal (intercaruncular stromal cell; ICAR) and epithelial origin. Both cell types are known to produce prostaglandins (e.g. PGF) and have immunomodulatory functions [8, 9]. Interactions between these cells may also play a pivotal role in endometrial receptivity during early pregnancy as was reported in a co-culture study that human endometrial stromal cells can mediate epithelial cell function by promoting differentiation and inhibiting proliferation of endometrial epithelial cells [10]. In the bovine, endometrial stromal cells (as utilized in the current study) are known to differentially regulate the production of prostaglandins and enzymes related to the production of prostaglandins, in response to specific stimuli (e.g. inflammatory mediators and interferon tau) [8, 11]. ICAR cells were a kind gift from Professor Michel A. Fortier (Université Laval, Québec). ICAR cells are a transformed cell-line derived from the intercaruncular region of the bovine endometrium [12]. ICAR cells can be propagated while still maintaining the phenotypical characteristics of these cells which include the presence of SV40 TAG and the vimentin-positive and cytokeratin-negative features that support the stromal phenotype of these cells [8, 13]. This study aimed to evaluate the effect of a known stimulus of exosome release on the production of exosomes by ICAR cells.

In recent years, evidence has emerged from studies in humans [14] and several other animal species [1518] that exosomes are secreted from the endometrium and trophoblast cells and may play important roles at the conceptus-endometrial interface during early pregnancy. Exosomes are specific subsets of extracellular vesicles (smaller than 1000 nm) [19] that could provide insights into an alternative new explanation for the crosstalk between cells. Exosomes (30–120 nm) are stable extracellular lipid bilayer vesicles arising from the inward budding of multivesicular bodies and released via an exocytic pathway to the extracellular environment with the capacity to modify the biological function of target cells [20]. Exosomes provide a mechanism of cell-to-cell communication in physiological and pathological conditions and may be influenced by neighboring cells, distant tissues or local environmental factors. There is considerable evidence that hypoxia is a potent stimulant to the release of exosomes [2124]. It is also a useful investigatory agent since a lower-than-normal oxygen tension in utero can influence many developmental events with potentially lifelong consequences [25, 26].

Hypoxia is a well-known stimulus of exosome release as seen in breast cancer cells, endothelial cells and human trophoblasts [24, 27, 28]. Alterations have been documented in both the number of exosomes released as well as differences in the content (cargo) of the exosomes [24, 27, 29]. This study aimed to test the hypothesis that hypoxia as a known stimulus of exosome release (and altered biological response) will modify the phenotype of bovine endometrial stromal cells affecting their migration, proliferation, apoptosis as well as altering both the release and cargo of the exosomes generated.

Methods

Aim

This study investigated the effect(s) of a hypoxic environment on the function of bovine endometrial cells. In particular, alterations to migration, proliferation and apoptosis. Moreover, this study evaluated alterations to the release and cargo content of exosomes generated by bovine endometrial cells, when cultured under hypoxia.

Endometrial cell line

A well characterized bovine endometrial intercaruncular stromal cell line (ICAR cells) was utilized for the current study [8, 30]. ICAR cells were a kind gift from Professor Michel A. Fortier (Université Laval, Québec). ICAR cells were maintained in 175 cm2 (T175, Corning Costar) culture flasks supplemented with exosome-free media (1640 Roswell Park Memorial Institute (RPMI) medium (Invitrogen, Life Technologies) with 10 % heat-inactivated fetal bovine serum (Bovogen, Interpath services Pty Ltd) depleted of exosomes by ultracentrifugation (100,000 g for 20 h at 4 °C) and 1000 U/mL antibiotic-antimycotic solution (Gibco, Life Technologies) in a humidified cell culture incubator at 37 °C under an atmosphere of 5 % CO2-balanced N2 to obtain a hypoxic (1 % O2) environment or under physiologically relevant conditions (8 % O2). Lactate dehydrogenase (LDH) assay was also performed accordingly to the manufacturer’s protocol using the commercially available kit Pierce LDH cytotoxicity assay kit (Thermo scientific) to measure LDH in supernatants of ICAR cells cultured at 8 % O2 and 1 % O2 and ICAR cell viability was accessed. No significant difference in the LDH activity was observed (data not shown) between 8 % O2 and 1 % O2, indicating that the viability of ICAR cells was not affected by experimental condition.

Cell migration assay

The effect of oxygen tension on cell migration was assessed using methods as previously published [31]. Briefly, ICAR cells were plated (30,000 cells per well) and grown to confluence in a 96-well culture plate (Corning Costar) at 1 % O2 or 8 % O2 oxygen tension and a wound scratch was made on confluent monolayers using a 96-pin WoundMaker (Essen BioScience). Migration assays were performed in the presence of Mitomycin C (100 ng/mL, Sigma–Aldrich) to minimize any confounding effects of cell proliferation. The wound images were automatically acquired every 2 h for 48 h and registered by the IncuCyte software system (Essen BioScience). Data are presented as the Relative Wound Density (RWD, Eizen, v1.0 algorithm). RWD is a representation of the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at every time point (time-curve).

Cell proliferation assay

Proliferation of ICAR cells was assessed using methods as previously published [28, 31]. In brief, the effect of oxygen tension on ICAR cell proliferation was assessed using a non-labelled cell monolayer confluence approach with a high density phase contrast real-time cell imaging system (IncuCyte™). ICAR cells were seeded at 40,000 cells per well in a 12-well culture plate (Corning Costar) and exposed to oxygen tension at 1 % O2 or 8 % O2 and the cell confluence (as the proliferation parameter) was measured at 0, 24 and 48 h.

Cell apoptosis assay

To assess the effect of hypoxia on cell apoptosis, ICAR cells were seeded at 5000 cells per well in 96-well culture plate (Corning Costar) in the presence of CellPlayer Kinetic Caspase-3/7 Apoptosis Assay Reagent (1:5000; Essen Biosciences) and imaged at 48 h with IncuCyte™. Cell apoptosis is determined by the measurement of the number of activated caspase 3/7 fluorescent objects count per mm2 divided by the percentage of cell confluence at 48 h (percentage of the area of field of view covered by cells with the metric ‘phase object confluence’) with the IncuCyte Zoom software using an integrated object counting algorithm.

Exosome isolation from cell-conditioned media

To study the effect of oxygen tension on exosome release, ICAR cells were incubated at 1 % O2 or 8 % O2 for 48 h. Exosomes were isolated from ICAR cell culture-conditioned media by successive differential centrifugation steps at 300 × g for 10 min and 2000 × g for 30 min. The supernatant was filtered through a 0.22-μm filter (Corning Costar) and ultracentrifuged at 100,000 × g for 20 h at 4 °C (Sorvall, SureSpin 630/360, Swinging-bucket ultracentrifuge rotor). Another round of ultracentrifugation washing steps was performed at 100,000 × g for 2 h at 4 °C (Beckman, Type 70.1 Ti, Fixed angle ultracentrifuge rotor). Exosomes were further enriched by layering on top of a discontinuous iodixanol gradient (OptiPrep, Sigma–Aldrich), which was centrifuged at 100,000 × g for 20 h (Beckman, Sw41Ti, Swinging-bucket ultracentrifuge rotor). Twelve fractions were obtained and diluted in 10 mL PBS (Gibco, Life Technologies). The fractions were washed with PBS and centrifuge at 100,000 × g for 2 h (Beckman, Type 70.1 Ti, Fixed angle ultracentrifuge rotor) and the exosomal pellets were suspended in 50 μL PBS.

Nanoparticle Tracking Analysis (NTA)

NTA measurements were performed using a NanoSight NS500 instrument (NanoSight NTA 3.0 Nanoparticle Tracking and Analysis Release Version Build 0064) as previously described [32, 33].

Western blot analysis and transmission electron microscopy

Exosomes were solubilized in RIPA buffer (Sigma–Aldrich) and separated by polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and probed with primary rabbit polyclonal antibody anti-CD63 (1:1000; EXOAB-CD63A-1, System Biosciences) and TSG101 (1:500; sc-6037, Santa Cruz Biotechnology). For electron microscopy analysis, exosome pellets were fixed in 3 % (w/v) glutaraldehyde and analyzed under an FEI Tecnai 12 transmission electron microscope (FEI, Hillsboro, Oregon, USA).

Proteomic Analysis of Endometrial Exosomes by Mass Spectrometry (MS)

Exosomes (10 μg of protein) were solubilized in RIPA buffer (Sigma–Aldrich) and separated by polyacrylamide gel electrophoresis. The gel was fixed in fixing solution (10:1:9; ethanol, acetic acid, MilliQ water respectively) for 15 min, washed in (1:1, ethanol and MilliQ water) for 10 min and washed three times with MilliQ water. Proteins were stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) for 1 h and the gel was allowed to destain in MilliQ water until a clear background was obtained.

In-gel digestion methods for the mass spectrometric identification of exosomal proteins were performed by modification of previously published method [34]. In brief, each sample lane was cut into 24 gel slices and destained twice with 200 mM ammonium bicarbonate in 50 % acetonitrile solution for 45 min at 37 °C, desiccated using a vacuum centrifuge and then resuspended with 20 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate solution and reduced for 1 h at 65 °C. DTT was then removed, and the samples were alkylated in 50 mM iodoacetamide and 25 mM ammonium bicarbonate at 37 °C in darkness for 40 min. Gel slices were washed three times for 45 min in 25 mM ammonium bicarbonate and then desiccated. Individual dried slices were then allowed to swell in 20 μL of 40 mM ammonium bicarbonate, 10 % acetonitrile containing 20 μg/mL trypsin (Sigma) for 1 h at room temperature. An additional 50 μL of the same solution was added and the samples were incubated overnight at 37 °C.

The supernatants were removed from the gel slices, and residual peptides were washed from the slices by incubating them three times in 50 μL of 0.1 % formic acid for 45 min at 37 °C. The original supernatant and washes were combined and desalted according to a modified version of the stage tip protocol that we have published [35, 36] using a 3-mm piece of an Empore C18 (Octadecyl) SPE Extraction Disk and the eluted peptides were dried in a vacuum centrifuge prior to spectral acquisition.

The digested protein samples were analysed using the TripleTOF® 5600 mass spectrometer (ABSciex, Redwood City, CA) and Eksigent 1D+ NanoLC system with the cHiPLC system to obtain initial high mass accuracy survey MS/MS data, identifying the peptides present in the samples. The ChromXP C18-CL TRAP cHiPLC (200 μm × 6 mm, 3 μm, 120 Å) and analytical cHiPLC columns (200 μm × 15 cm; 3 μm, 120 Å) (Eksigent, Redwood City, CA) were used to separate the digested proteins. A 10 μL aliquot of digested material was injected onto the column and separated with a linear gradient of 5 to 10 % Buffer B for 2 min (Buffer A: 0.1 % Formic acid/water; Buffer B: acetonitrile/0.1 % formic acid), 10 to 40 % Buffer B (58 min), 40 to 50 % Buffer B (10 min), 50 to 95 % (10 min) with a flow rate of 500 nL/min. The column was flushed at 95 % buffer B for 15 min and re-equilibrated with 5 % Buffer B for 6 min. The in-depth proteomic analysis was performed using the Information Dependent Acquisition (IDA) experiments on the TripleTOF® 5600 System interfaced with a nanospray source. The source parameters were as follows: Cur gas at 25 psi, GS1 at 5 psi and IHT at 150 °C. A 250 msec accumulation time was set for the TOFMS survey scan and from this scan, the 10 most intense precursor ions were selected automatically for the MS/MS analysis (accumulation time of 150 msecs per MS/MS scan). Ions were isolated using unit resolution of the quadrupoles and rolling collision energy equation was used to calculate the collision energies of precursors. The precursor selection criteria included a minimum intensity of 50 counts per second (cps) and a charge state greater than 2 + .

Protein identification was determined using the ProteinPilot™ Software (v4.5 beta, AB Sciex, Redwood City, CA) with the Paragon algorithm. The search parameters were as follows: sample type, identification; cys alkylation, iodoacetamide; digestion, Trypsin; Instrument, TripleTOF 5600; special factors, none; and ID focus, biological modifications. The database was downloaded from the UniProt website in October 2015, which contained all proteins from Bos taurus. False discovery rate (FDR) was selected in the method and determined using a reversed sequence database. Data were subjected to ontology and pathway analysis using the protein analysis through evolutionary relationships tool (PANTHER) and gene ontology algorithms and classified based on biological process and molecular function categories [37].

Statistical analyses

The effects of oxygen tensions on ICAR cells are presented as mean ± SE for migration, proliferation and apoptosis assays (n = 6 independent experiments in duplicate). The number of exosomes is presented as number of particles per mL (mean ± SE, n = 3 independent isolations from 80 million cells each). The effects of oxygen tension on ICAR cells were identified by Student’s T tests (two-tailed) to compare the effect of hypoxia (i.e. 1 % O2) with the control group (i.e. 8 % O2) using a commercially-available software package (Prism 6, GraphPad Inc, La Jolla, CA 92037 USA).

Results

The Effect of Oxygen Tension on Bovine Endometrial (ICAR) cell migration and proliferation

The effect of normal oxygen tension (i.e. 8 % O2) and hypoxia (i.e. 1 % O2) on ICAR cell migration is presented in Fig. 1. ICAR cell migration was significantly lower under hypoxia compared with normal oxygen tension (Fig. 1a). Hypoxia decreased ICAR cell migration in a time-dependent manner (Fig. 1b). Area under the curve analysis indicated that hypoxia decreased ICAR cell migration by ~20 % compared with values observed at 8 % O2 (2173 ± 36 and 2620 ± 50 for 1 % O2 and 8 % O2, respectively) (Fig. 1c). Interestingly, hypoxia decreased ICAR cell proliferation in a time-dependent manner (Fig. 2a and b). Area under curve analysis showed that at 1 % O2, the proliferative capacity of ICAR cells was inhibited (p < 0.05) ~32 % compared with cell proliferation at 8 % O2 (2.32 ± 0.18 and 3.41 ± 0.2 for 1 % O2 and 8 % O2, respectively) (Fig. 2c).
Fig. 1

The effects of different oxygen tension on migration of bovine endometrial stromal cells (ICAR). a Graphical representation of the initial wound width (white) at 0 h and the area of the initial wound covered by advancing cells (grey) at 24 h and 48 h, Scale bar 300 μm. b Decreased ICAR cell migration under hypoxic conditions (1 % O2 () compared with a normoxic 8 % O2 ()) over a period of 48 h. c Area under the curve analysis from (b); 8 % O2 (white bar) and 1 % O2 (black bar). Data are presented as mean ± SE, n = 6. In (b) and (c) P < 0.05

Fig. 2

The effects of different oxygen tension on proliferation of bovine endometrial stromal cells (ICAR). a Representative phase-contrast image of ICAR cells at 48 h when cultured under hypoxic conditions (1 % O2) compared with a normoxic 8 % O2, Scale bar 200 μm. b Decreased (p < 0.01) ICAR cell proliferation under hypoxic conditions (1 % O2 ()) compared with a normoxic 8 % O2 () over a period of 48 h. c Area under the curve analysis from (b); 8 % O2 (white bar) and 1 % O2 (black bar). Data are presented as mean ± SE, n = 6. In (C) P < 0.05

The Effect of Oxygen Tension on Bovine Endometrial (ICAR) cell apoptosis

The effect of oxygen tension on cell apoptosis is presented in Fig. 3. A hypoxic (1 % O2) environment altered cell morphology compared with cells cultured under normal conditions (8 % O2), displaying morphological hallmarks of apoptotic death (Fig. 3A ,a and d). Fluorescent images acquired with IncuCyte™ (Fig. 3A, b and e) showed greater fluorescence in cells cultured under 1 % O2, indicating a higher activation of caspase-3/7 under hypoxic conditions compared with 8 % O2 (Fig. 3A, b and e). Apoptosis was quantified using the object counting algorithm in which the number of fluorescent objects was indicated with red x’s in Fig. 3A (c and f). Quantification analysis showed that hypoxia increased (~1.6 fold) the apoptosis ratio (presented as activated caspase 3/7 fluorescent objects count per mm2 divided by percentage of cell confluence at 48 h) compared with cells cultured under normal oxygen tension (Fig. 3B).
Fig. 3

The effects of different oxygen tension on activation of apoptotic protein caspase-3 of bovine endometrial stromal cells (ICAR). ICAR cells were cultured under normoxic (8 % O2) or hypoxic (1 % O2) conditions and the activated caspase-3/7 fluorescence was measured at 48 h. A Representative phase-contrast images (a and d), fluorescent signal images (b and e) and acquired fluorescent signal using integrated object counting algorithm with IncuCyte™ (Segmentation; c and f), Scale bar 400 μm. B Increased apoptosis of ICAR cells under hypoxic conditions as determined by acquired fluorescent signal using integrated object counting algorithm with IncuCyte™ were normalized against cell confluence, 8 % O2 (white bar) and 1 % O2 (black bar). Data are presented as mean ± SE, n = 6. In (B) P < 0.05

The Effect of Oxygen Tension on Exosome Release from Bovine Endometrial Cells (ICAR)

Exosomes were enriched by buoyant density gradient (see Material and Methods). We fractioned the 100,000 × g pellet into 12 fractions and the Western blot analysis for TSG101 and CD63 showed positive protein abundance in fractions 1.17 and 1.18 g/mL (Fig. 4a). Exosomes were pooled between densities 1.16 and 1.18 g/mL. Morphology of exosomes was determined by electron microscopy (Fig. 4b), exosomes displayed a cup-shaped morphology with an estimated diameter of 100 nm. Hypoxia did not alter the size distribution of exosomes compared with normal oxygen tension (123 ± 2.7 nm versus 127 ± 1.7 nm for 8 % O2 and 1 % O2, respectively) (Fig. 4c). Interestingly, hypoxia increased (~3.6 fold) the number of exosomes compared with values observed at normal oxygen tension (Fig. 4d).
Fig. 4

Characterization of exosomes release from 8 % O2 and 1 % O2 ICAR cell-conditioned media. Exosomes were characterized after enrichment from the 100,000 x g pellet by buoyant density centrifugation (see Methods). a Representative Western blot for exosome markers: TSG101 and CD63. b Representative electron micrograph exosome fractions, Scale bar 100 nm. c Representative Nanosight measurement of particle-size distribution exosomes from 8 % O2 and 1 % O2 cell-conditioned media after buoyant density gradient ultracentrifugation. (8 % normoxic condition mean size (127 ± 1.7 nm) (), 1 % hypoxic condition mean size (123 ± 2.7 nm) () over a period of 48 h). d Exosomes concentration presented as vesicle per million cells per 48 h was higher (p < 0.05) at hypoxia (1 % O2) compared to normal oxygen tension (8 % O2); 8 % O2 (white bar) and 1 % O2 (black bar). Data are presented as mean ± SE, n = 3

Proteomic Analysis of Bovine Endometrial ICAR-Derived Exosomes

Mass spectrometric analysis identified over 250 exosomal proteins with 113 similar proteins identified as present in both exosomes of ICAR cultured at 1 % O2 and at 8 % O2 128 proteins identified as unique to exosomes of ICAR cultured at 1 % O2; 46 proteins were identified as unique to exosomes of ICAR cultured at 8 % O2 (Table 1 A-C; Fig. 5a). Data were subjected to ontology and pathway analysis using PANTHER and gene ontology algorithms and classified based on biological process (Fig. 5b) and molecular function (Fig. 5c). In biological process, the clusters identified from individual proteins that are unique to and present only in exosomes of ICAR cultured at 1 % O2 but not those at 8 % O2 were: growth (0.7 %), locomotion (0.7 %) and reproduction (1.4 %) (Fig. 5b). In molecular functions, the proteins related to binding and catalytic activity were the greatest recognized in both exosomes of ICAR cultured at 1 % O2 and to those of ICAR cultured at 8 % O2 (Fig. 5c).
Table 1

List of the common proteins identified in exosomes of ICAR cultured at 1 % O2 and at 8 % O2

A. List of 113 common proteins identified in exosomes of ICAR cultured at 1 % O2 and at 8 % O2

Protein ID

Name

Gene Name

Biological Process (Total # Gene 69; Total #Function 146)

Molecular function (Total # Gene 69; Total #Function 81)

 A1L523_BOVIN

Copine II (Fragment)

CPNE2

  

 A3KN51_BOVIN

TSG101 protein

TSG101

Metabolic process

Catalytic activity

 A5D7L1_BOVIN

CLEC11A protein

CLEC11A

Cellular process/Developmental process

Binding/Structural molecule activity

 A5D9D2_BOVIN

Complement component 4 binding protein, alpha chain

C4BPA

  

 A5PJ69_BOVIN

SERPINA10 protein

SERPINA10

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 A5PJE3_BOVIN

Fibrinogen alpha chain

FGA

  

 A5PK77_BOVIN

SERPINA11 protein

SERPINA11

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 A6QLB7_BOVIN

Adenylyl cyclase-associated protein

CAP1

  

 A6QLL8_BOVIN

Fructose-bisphosphate aldolase

ALDOA

  

 A6QNZ7_BOVIN

Keratin 10 (Epidermolytic hyperkeratosis; keratosis palmaris et plantaris)

KRT10

  

 A6QPP2_BOVIN

SERPIND1 protein

SERPIND1

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 A6QPR1_BOVIN

PCYOX1 protein

PCYOX1

  

 LG3BP_BOVIN

Galectin-3-binding protein

LGALS3BP

Apoptotic process/Biological adhesion/Biological regulation/Cellular process/Developmental process/Immune system process/localization/Metabolic process

Catalytic activity/Receptor activity

 A7MB82_BOVIN

C1QTNF3 protein

C1QTNF3

  

 A7YWB6_BOVIN

LOC539596 protein

LOC539596

  

 B0JYM4_BOVIN

Tetraspanin

CD63

  

 B0JYN6_BOVIN

Alpha-2-HS-glycoprotein

AHSG

  

 B0JYQ0_BOVIN

ALB protein

ALB

  

 B5B3R8_BOVIN

Alpha S1 casein

CSN1S1

  

 E1BDG5_BOVIN

Protein Wnt

WNT5A

Biological regulation/Cellular process/Developmental process/Multicellular organismal process/Response to stimulus

Binding

 CBG_BOVIN

Corticosteroid-binding globulin

SERPINA6

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 F1MAV0_BOVIN

Fibrinogen beta chain

FGB

  

 F1MB08_BOVIN

Alpha-enolase

ENO1

  

 F1MC11_BOVIN

Keratin, type I cytoskeletal 14

KRT14

  

 F1MM32_BOVIN

Sulfhydryl oxidase

QSOX1

 

Catalytic activity

 F1MMK9_BOVIN

Protein AMBP

AMBP

  

 F1MMP5_BOVIN

Inter-alpha-trypsin inhibitor heavy chain H1

ITIH1

  

 ITA3_BOVIN

Integrin alpha-3

ITGA3

  

 F1MNW4_BOVIN

Inter-alpha-trypsin inhibitor heavy chain H2

ITIH2

  

 F1MSZ6_BOVIN

Antithrombin-III

SERPINC1

  

 F1MTV5_BOVIN

Amino acid transporter

SLC1A5

  

 F1MW44_BOVIN

Coagulation factor XIII A chain

F13A1

  

 F1MXJ5_BOVIN

IST1 homolog

IST1

  

 F1MXX6_BOVIN

Lactadherin

MFGE8

  

 F1MY85_BOVIN

Complement C5a anaphylatoxin

C5

  

 F1N045_BOVIN

Complement component C7

C7

  

 HTRA1_BOVIN

Serine protease HTRA1

HTRA1

Cellular process/Metabolic process

Catalytic activity

 F1N1I6_BOVIN

Gelsolin

GSN

  

 F6QVC9_BOVIN

Annexin

ANXA5

  

 G3X6N3_BOVIN

Serotransferrin

TF

  

 G5E5A9_BOVIN

Fibronectin

FN1

  

 G5E5V0_BOVIN

Carboxypeptidase N catalytic chain

CPN1

  

 G8JKX6_BOVIN

Tetraspanin (Fragment)

CD9

  

 I7CT57_BOVIN

Vitamin D binding protein

   

 M0QVZ6_BOVIN

Keratin, type II cytoskeletal 5

KRT5

  

 THRB_BOVIN

Prothrombin

F2

Immune system process/Metabolic process/Response to stimulus

Catalytic activity

 PROC_BOVIN

Vitamin K-dependent protein C (Fragment)

PROC

Response to stimulus

Binding

 KNG2_BOVIN

Kininogen-2

KNG2

  

 THYG_BOVIN

Thyroglobulin

TG

Metabolic process

Catalytic activity

 HBA_BOVIN

Hemoglobin subunit alpha

HBA

localization/Multicellular organismal process

 

 HBBF_BOVIN

Hemoglobin fetal subunit beta

 

localization/Multicellular organismal process

 

 ALBU_BOVIN

Serum albumin

ALB

localization

 

 ANXA2_BOVIN

Annexin A2

ANXA2

Developmental process/Metabolic process

 

 ASSY_BOVIN

Argininosuccinate synthase

ASS1

Cellular process/Metabolic process

Catalytic activity

 APOH_BOVIN

Beta-2-glycoprotein 1

APOH

Cellular process/Immune system process/localization/Metabolic process/Response to stimulus

Catalytic activity/Receptor activity/Transporter activity

 CLUS_BOVIN

Clusterin

CLU

  

 HSP7C_BOVIN

Heat shock cognate 71 kDa protein

HSPA8

Cellular component organization or biogenesis/Immune system process/Metabolic process/Response to stimulus

 

 ANXA7_BOVIN

Annexin A7

ANXA7

Metabolic process

 

 ANX11_BOVIN

Annexin A11

ANXA11

Metabolic process

 

 A2AP_BOVIN

Alpha-2-antiplasmin

SERPINF2

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 A1AT_BOVIN

Alpha-1-antiproteinase

SERPINA1

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 GDIB_BOVIN

Rab GDP dissociation inhibitor beta

GDI2

Biological regulation/Cellular process/localization/Metabolic process/Multicellular organismal process

Binding/Catalytic activity/Enzyme regulator activity

 F12AI_BOVIN

Factor XIIa inhibitor

   

 ITB1_BOVIN

Integrin beta-1

ITGB1

Biological adhesion/Cellular process/Response to stimulus

Receptor activity

 ITIH3_BOVIN

Inter-alpha-trypsin inhibitor heavy chain H3

ITIH3

Biological regulation/Metabolic process

Binding/Catalytic activity/Enzyme regulator activity

 ACTB_BOVIN

Actin, cytoplasmic 1

ACTB

Cellular component organization or biogenesis/Cellular process/Developmental process/localization

Structural molecule activity

 ANXA6_BOVIN

Annexin A6

ANXA6

Metabolic process

 

 CFAB_BOVIN

Complement factor B

CFB

Biological adhesion/Cellular process/Immune system process/localization/Metabolic process/Response to stimulus

Catalytic activity/Receptor activity/Transporter activity

 TBA1B_BOVIN

Tubulin alpha-1B chain

 

Cellular process/Developmental process/localization

Structural molecule activity

 LUM_BOVIN

Lumican

LUM

Biological adhesion/Biological regulation/Cellular process/Developmental process/Immune system process/Metabolic process/Multicellular organismal process

Receptor activity

 UPAR_BOVIN

Urokinase plasminogen activator surface receptor

PLAUR

  

 5NTD_BOVIN

5’-nucleotidase

NT5E

Metabolic process

Catalytic activity

 PGM1_BOVIN

Phosphoglucomutase-1

PGM1

Cellular process/Metabolic process

Catalytic activity

 Q09TE3_BOVIN

Insulin-like growth factor binding protein acid labile subunit

   

 Q17R18_BOVIN

Adenosine kinase

ADK

  

 FA5_BOVIN

Coagulation factor V

F5

Biological adhesion/Biological regulation/Cellular process/Developmental process/Immune system process/localization/Metabolic process/Multicellular organismal process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity/Transporter activity

 Q2KIF2_BOVIN

Leucine-rich alpha-2-glycoprotein 1

LRG1

Cellular process/Multicellular organismal process

Receptor activity

 CBPB2_BOVIN

Carboxypeptidase B2

CPB2

Metabolic process

Catalytic activity

 Q2KJ47_BOVIN

EH-domain containing 2

EHD2

Biological regulation/Cellular process/localization/Metabolic process/Multicellular organismal process

Binding/Catalytic activity/Enzyme regulator activity

 TBB5_BOVIN

Tubulin beta-5 chain

TUBB5

Cellular process/Developmental process/localization

Structural molecule activity

 A1BG_BOVIN

Alpha-1B-glycoprotein

A1BG

Cellular process/Immune system process/Response to stimulus

Binding/Receptor activity

 HPT_BOVIN

Haptoglobin

HP

Biological regulation/Immune system process/localization/Metabolic process/Multicellular organismal process/Reproduction/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity

 CO3_BOVIN

Complement C3

C3

Biological regulation/Cellular process/Metabolic process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity

 Q3MHH8_BOVIN

Alpha-amylase

AMY2A

  

 SAHH_BOVIN

Adenosylhomocysteinase

AHCY

Cellular process/Metabolic process

Catalytic activity

 CO9_BOVIN

Complement component C9

C9

Cellular process/localization/Metabolic process/Response to stimulus

Catalytic activity/Receptor activity/Transporter activity

 Q3MHW2_BOVIN

F10 protein (Fragment)

F10

  

 Q3MHZ0_BOVIN

FLOT1 protein (Fragment)

FLOT1

  

 Q3SYR0_BOVIN

Serpin peptidase inhibitor, clade A (Alpha-1 antiproteinase, antitrypsin), member 7

SERPINA7

  

 FETA_BOVIN

Alpha-fetoprotein

AFP

Developmental process/localization

 

 Q3SZH5_BOVIN

Angiotensinogen

AGT

  

 HEMO_BOVIN

Hemopexin

HPX

localization

 

 Q3SZZ9_BOVIN

FGG protein

FGG

  

 PGK1_BOVIN

Phosphoglycerate kinase 1

PGK1

Metabolic process

Catalytic activity

 Q3T101_BOVIN

IGL@ protein

IGL@

  

 G6PI_BOVIN

Glucose-6-phosphate isomerase

GPI

Metabolic process

Catalytic activity

 Q3ZBX0_BOVIN

Basigin

BSG

  

 Q3ZC87_BOVIN

Pyruvate kinase (Fragment)

PKM2

  

 Q3ZCI4_BOVIN

6-phosphogluconate dehydrogenase, decarboxylating

PGD

Metabolic process

Catalytic activity

 FETUB_BOVIN

Fetuin-B

FETUB

  

 EHD1_BOVIN

EH domain-containing protein 1

EHD1

Biological regulation/Cellular process/localization/Metabolic process/Multicellular organismal process

Binding/Catalytic activity/Enzyme regulator activity

 HPPD_BOVIN

4-hydroxyphenylpyruvate dioxygenase

HPD

Metabolic process

Catalytic activity

 Q5EA67_BOVIN

Inter-alpha (Globulin) inhibitor H4 (Plasma Kallikrein-sensitive glycoprotein)

ITIH4

  

 Q5GN72_BOVIN

Alpha-1-acid glycoprotein

agp

  

 BHMT1_BOVIN

Betaine--homocysteine S-methyltransferase 1

BHMT

Cellular process/Metabolic process

Catalytic activity

 Q5J801_BOVIN

Endopin 2B

   

 Q6T182_BOVIN

Sex hormone-binding globulin (Fragment)

SHBG

  

 A2MG_BOVIN

Alpha-2-macroglobulin

A2M

Biological regulation/Cellular process/Immune system process/Metabolic process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity

 PEDF_BOVIN

Pigment epithelium-derived factor

SERPINF1

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 CHIA_BOVIN

Acidic mammalian chitinase

CHIA

Immune system process/Metabolic process/Response to stimulus

Binding/Catalytic activity

 IPSP_BOVIN

Plasma serine protease inhibitor

SERPINA5

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 SPA31_BOVIN

Serpin A3-1

SERPINA3-1

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 V6F9A2_BOVIN

Apolipoprotein A-I preproprotein

APOA1

  

B. List of 128 unique proteins identified in exosomes of ICAR cultured at 1 % O2

Protein ID

Name

Gene Name

Biological Process (Total # Gene 22; Total #Function 49)

Molecular function (Total # Gene 22; Total #Function 28)

 G3X6T9_BOVIN

Flotillin-2 (Fragment)

FLOT2

  

 TSP1_BOVIN

Thrombospondin-1

THBS1

  

 F1N2L9_BOVIN

4-trimethylaminobutyraldehyde dehydrogenase

ALDH9A1

  

 E1B9F6_BOVIN

Elongation factor 1-alpha

EEF1A1

  

 APOE_BOVIN

Apolipoprotein E

APOE

Apoptotic process/Biological regulation/Cellular component organization or biogenesis/Cellular process/Developmental process/Growth/localization/Metabolic process/Multicellular organismal process/Response to stimulus

Binding/Catalytic activity/ Enzyme regulator activity/Transporter activity

 G1K1R6_BOVIN

Galactokinase

GALK1

  

 G3P_BOVIN

Glyceraldehyde-3-phosphate dehydrogenase

GAPDH

Metabolic process

Catalytic activity

 Q0P5B0_BOVIN

Arrestin domain containing 1

ARRDC1

  

 RL40_BOVIN

Ubiquitin-60S ribosomal protein L40

UBA52

Metabolic process

Binding/Structural molecule activity

 A5D9B6_BOVIN

Syntenin

SDCBP

  

 Q8HZY1_BOVIN

Serine protease inhibitor clade E member 2

SERPINE2

  

 Q5E962_BOVIN

Aldo-keto reductase family 1, member B1

AKR1B1

  

 A7MBH9_BOVIN

GNAI2 protein

GNAI2

Biological regulation/Cellular process/Metabolic process/Response to stimulus

Binding/Catalytic activity

 GBB2_BOVIN

Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2

GNB2

Cellular process/Metabolic process/Multicellular organismal process

Binding/Catalytic activity

 I6YIV1_BOVIN

Annexin

   

 F16P1_BOVIN

Fructose-1,6-bisphosphatase 1

FBP1

Metabolic process

 

 F1N3Q7_BOVIN

Apolipoprotein A-IV

APOA4

  

 AK1A1_BOVIN

Alcohol dehydrogenase [NADP(+)]

AKR1A1

localization/Metabolic process

Catalytic activity/Transporter activity

 A5D784_BOVIN

CPNE8 protein

CPNE8

localization

 

 HS90A_BOVIN

Heat shock protein HSP 90-alpha

HSP90AA1

Immune system process/Metabolic process/Response to stimulus

 

 Q1JPA2_BOVIN

Eukaryotic translation elongation factor 1 gamma (Fragment)

EEF1G

  

 SERA_BOVIN

D-3-phosphoglycerate dehydrogenase

PHGDH

Metabolic process

Catalytic activity

 Q3T085_BOVIN

OGN protein

OGN

  

 A8DBT6_BOVIN

Monocyte differentiation antigen CD14

CD14

  

 A5PK73_BOVIN

Fructose-bisphosphate aldolase

ALDOB

  

 G5E5U7_BOVIN

S-adenosylmethionine synthase

MAT1A

  

 F1N2W0_BOVIN

Prostaglandin reductase 1

PTGR1

  

 IF4A1_BOVIN

Eukaryotic initiation factor 4A-I

EIF4A1

Biological regulation/Metabolic process

Binding/Catalytic activity/Translation regulator activity

 Q05B55_BOVIN

IGK protein

IGK

  

 F1N1D4_BOVIN

Protein tweety homolog

TTYH3

localization

Transporter activity

 A4FV94_BOVIN

KRT6A protein

KRT6A

  

 RGN_BOVIN

Regucalcin

RGN

Cellular process/localization/Metabolic process

Binding/Catalytic activity

 1433E_BOVIN

14-3-3 protein epsilon

YWHAE

Cellular process

 

 Q2HJB6_BOVIN

Procollagen C-endopeptidase enhancer

PCOLCE

Biological adhesion/Biological regulation/Cellular process/Developmental process/Immune system process/localization/Metabolic process/Multicellular organismal process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity/Transporter activity

 B8YB76_BOVIN

Homogentisate 1,2-dioxygenase

HGD

  

 DHSO_BOVIN

Sorbitol dehydrogenase

SORD

Metabolic process

Catalytic activity

 HS71A_BOVIN

Heat shock 70 kDa protein 1A

HSPA1A

Cellular component organization or biogenesis/Immune system process/Metabolic process/Response to stimulus

 

 Q3ZBQ9_BOVIN

APOM protein

APOM

  

 PYGL_BOVIN

Glycogen phosphorylase, liver form

PYGL

Metabolic process

Catalytic activity

 A6QP30_BOVIN

CPN2 protein

CPN2

Cellular process/Multicellular organismal process

Receptor activity

 ARF3_BOVIN

ADP-ribosylation factor 3

ARF3

Cellular process/localization/Metabolic process

Binding/Catalytic activity

 G3MYH4_BOVIN

Tetraspanin (Fragment)

CD81

  

 ACTC_BOVIN

Actin, alpha cardiac muscle 1

ACTC1

Cellular component organization or biogenesis/Cellular process/Developmental process/localization

Structural molecule activity

 GALM_BOVIN

Aldose 1-epimerase

GALM

Metabolic process

Catalytic activity

 TSN6_BOVIN

Tetraspanin-6

TSPAN6

Biological adhesion/Cellular process/Immune system process/Multicellular organismal process/Reproduction/Response to stimulus

Binding/Receptor activity

 Q3ZC83_BOVIN

Solute carrier family 29 (Nucleoside transporters), member 1

SLC29A1

localization/Metabolic process

Transporter activity

 B4GA1_BOVIN

Beta-1,4-glucuronyltransferase 1

B4GAT1

Metabolic process

Catalytic activity

 ADA10_BOVIN

Disintegrin and metalloproteinase domain-containing protein 10

ADAM10

Apoptotic process/Developmental process/Reproduction

 

 A6QR28_BOVIN

Phosphoserine aminotransferase

PSAT1

Metabolic process

Catalytic activity

 Q1JPB6_BOVIN

Acetyl-Coenzyme A acetyltransferase 2

ACAT2

  

 DDBX_BOVIN

Dihydrodiol dehydrogenase 3

 

localization/Metabolic process

Catalytic activity/Transporter activity

 A2VE11_BOVIN

IGSF8 protein

IGSF8

  

 F1MS32_BOVIN

Apolipoprotein D

APOD

  

 A6QP64_BOVIN

VPS37B protein (Fragment)

VPS37B

  

 Q2KIW4_BOVIN

Lecithin-cholesterol acyltransferase

LCAT

Metabolic process

Catalytic activity

 GBB1_BOVIN

Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

GNB1

Cellular process/Metabolic process

Binding/Catalytic activity

 GNA11_BOVIN

Guanine nucleotide-binding protein subunit alpha-11

GNA11

Biological regulation/Cellular process/Metabolic process/Response to stimulus

Catalytic activity

 Q17QK4_BOVIN

Epoxide hydrolase 2, cytoplasmic

EPHX2

  

 K2C7_BOVIN

Keratin, type II cytoskeletal 7

KRT7

Cellular component organization or biogenesis/Cellular process/Developmental process

Structural molecule activity

 CLIC1_BOVIN

Chloride intracellular channel protein 1

CLIC1

Biological regulation/Cellular process/Metabolic process/Response to stimulus

Binding/Catalytic activity/Structural molecule activity/Translation regulator activity

 Q08DW4_BOVIN

Mannan-binding lectin serine peptidase 1 (C4/C2 activating component of Ra-reactive factor)

MASP1

  

 B4GT1_BOVIN

Beta-1,4-galactosyltransferase 1

B4GALT1

  

 A5D7E6_BOVIN

Tetraspanin

CD82

Cellular process/Response to stimulus

Binding/Receptor activity

 A5D973_BOVIN

Alpha isoform of regulatory subunit A, protein phosphatase 2

PPP2R1A

  

 E1B726_BOVIN

Plasminogen

PLG

  

 G5E6I9_BOVIN

Histone H2B

LOC101904777

Cellular component organization or biogenesis/Cellular process/Metabolic process

Binding

 ADIPO_BOVIN

Adiponectin

ADIPOQ

  

 F1MBC5_BOVIN

Coagulation factor IX

F9

  

 A2VDL2_BOVIN

Solute carrier family 2 (Facilitated glucose transporter), member 3

SLC2A3

  

 VPS4B_BOVIN

Vacuolar protein sorting-associated protein 4B

VPS4B

  

 G3X8B1_BOVIN

Peptidyl-prolyl cis-trans isomerase

LOC613401

  

 K4JB97_BOVIN

Alpha-2-macroglobulin variant 4

A2M

  

 ACTG_BOVIN

Actin, cytoplasmic 2

ACTG1

Cellular component organization or biogenesis/Cellular process/localization

Structural molecule activity

 Q1JPG7_BOVIN

Pyruvate kinase

PKLR

  

 GTR1_BOVIN

Solute carrier family 2, facilitated glucose transporter member 1

SLC2A1

  

 F1N342_BOVIN

Protein tweety homolog

TTYH2

localization

Transporter activity

 ADHX_BOVIN

Alcohol dehydrogenase class-3

ADH5

Metabolic process

Catalytic activity

 URP2_BOVIN

Fermitin family homolog 3

FERMT3

  

 E1B7N2_BOVIN

Histone H4

HIST1H4I

Cellular component organization or biogenesis/Cellular process/Metabolic process

Binding

 EF2_BOVIN

Elongation factor 2

EEF2

Biological regulation/Metabolic process

Binding/Translation regulator activity

 KLKB1_BOVIN

Plasma kallikrein

KLKB1

Biological regulation/localization/Metabolic process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity

 ESTD_BOVIN

S-formylglutathione hydrolase

ESD

Metabolic process

Catalytic activity

 SEPR_BOVIN

Prolyl endopeptidase FAP

FAP

Cellular process/Immune system process/localization/Metabolic process/Multicellular organismal process / Response to stimulus

Binding/Catalytic activity

 Q5EA54_BOVIN

Solute carrier family 3 (Activators of dibasic and neutral amino acid transport), member 2

SLC3A2

  

 Q1JPD9_BOVIN

G protein-coupled receptor, family C, group 5, member B

GPRC5B

Cellular process

Receptor activity

 F1MS05_BOVIN

Aconitate hydratase

ACO1

  

 F1MJ12_BOVIN

Complement C1s subcomponent

C1S

  

 CNDP2_BOVIN

Cytosolic non-specific dipeptidase

CNDP2

Metabolic process

Catalytic activity

 Q2TBQ1_BOVIN

Coagulation factor XIII, B polypeptide

F13B

Biological adhesion/Cellular process/Immune system process/localization/Metabolic process/Response to stimulus

Catalytic activity/Receptor activity/Transporter activity

 Q1JP72_BOVIN

Colony stimulating factor 1 receptor

CSF1R

  

 Q0VD03_BOVIN

CD44 antigen

CD44

  

 G3X6Y4_BOVIN

Osteomodulin

OMD

  

 GAMT_BOVIN

Guanidinoacetate N-methyltransferase

GAMT

  

 VWA1_BOVIN

von Willebrand factor A domain-containing protein 1

VWA1

  

 SERC3_BOVIN

Serine incorporator 3

SERINC3

  

 Q862H8_BOVIN

Similar to 40S ribosomal protein SA (P40) (Fragment)

   

 A8E4P3_BOVIN

STOM protein

STOM

  

 F1MHP6_BOVIN

Adenylosuccinate lyase

ADSL

  

 E1BMG9_BOVIN

10-formyltetrahydrofolate dehydrogenase

ALDH1L1

Metabolic process

Catalytic activity

 Q705V4_BOVIN

Kappa-casein (Fragment)

csn3

  

 G3X6Q8_BOVIN

Pentraxin-related protein PTX3

PTX3

  

 K7QEL2_BOVIN

MHC class I antigen

BoLA

  

 TCPQ_BOVIN

T-complex protein 1 subunit theta

CCT8

Cellular component organization or biogenesis / Metabolic process

 

 F1N6Z0_BOVIN

26S proteasome non-ATPase regulatory subunit 5

PSMD5

  

 ARLY_BOVIN

Argininosuccinate lyase

ASL

Metabolic process

Catalytic activity

 E1BNG2_BOVIN

alpha-1,2-Mannosidase

MAN1A1

Metabolic process

 

 F1MU79_BOVIN

Peptidyl-prolyl cis-trans isomerase FKBP4

FKBP4

  

 DPYL2_BOVIN

Dihydropyrimidinase-related protein 2

DPYSL2

Metabolic process

Catalytic activity

 PRS23_BOVIN

Serine protease 23

PRSS23

  

 B0JYN1_BOVIN

Cathepsin L2

CTSL2

  

 A4FV99_BOVIN

FCNB protein

FCNB

  

 A7YW37_BOVIN

CD58 protein (Fragment)

CD58

Immune system process/Response to stimulus

Binding

 F1MTP5_BOVIN

WD repeat-containing protein 1

WDR1

  

 A7E3D0_BOVIN

CCDC45 protein (Fragment)

CCDC45

  

 Q0VCK1_BOVIN

Myeloid-associated differentiation marker

MYADM

  

 A1L570_BOVIN

Ephrin-B1

EFNB1

Biological regulation/Cellular component organization or biogenesis/Cellular process/Developmental process/locomotion/Multicellular organismal process/Response to stimulus

Binding

 F1N049_BOVIN

Actin-related protein 3 (Fragment)

ACTR3

  

 PAI1_BOVIN

Plasminogen activator inhibitor 1

SERPINE1

Biological regulation/Metabolic process

Catalytic activity/Enzyme regulator activity

 Q3ZC30_BOVIN

Sulfotransferase

SULT1E1

  

 COL11_BOVIN

Collectin-11

COLEC11

Biological regulation/Immune system process/Multicellular organismal process

 

 MPZL1_BOVIN

Myelin protein zero-like protein 1

MPZL1

Cellular process/localization

Transporter activity

 G5E595_BOVIN

Lys-63-specific deubiquitinase BRCC36

BRCC3

  

 O18977_BOVIN

Tenascin-X

TN-X

  

 A6H7D3_BOVIN

KRT18 protein (Fragment)

KRT18

  

 J9ZXG5_BOVIN

Integrin alpha V subunit

   

 B0JYN3_BOVIN

L-lactate dehydrogenase

LDHB

  

 MB211_BOVIN

Protein mab-21-like 1

MAB21L1

  

 E1B7R4_BOVIN

Eukaryotic translation initiation factor 3 subunit A

EIF3A

Biological regulation/Metabolic process

Binding/Translation regulator activity

C. List of 46 unique proteins identified in exosomes of ICAR cultured at 8 % O2

Protein ID

Name

Gene Name

Biological Process (Total # Gene 22; Total #Function 49)

Molecular function (Total # Gene 22; Total #Function 28)

 F1MMD7_BOVIN

Inter-alpha-trypsin inhibitor heavy chain H4

ITIH4

  

 F1N3A1_BOVIN

Thrombospondin-1

THBS1

  

 PLMN_BOVIN

Plasminogen

PLG

Biological regulation/localization/Metabolic process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity

 F1MYN5_BOVIN

Fibulin-1

FBLN1

Cellular process/Developmental process

Binding

 F1MNV5_BOVIN

Kininogen-1

KNG1

  

 EF1A1_BOVIN

Elongation factor 1-alpha 1

EEF1A1

Biological regulation/Metabolic process

Binding/Catalytic activity/Translation regulator activity

 ITAV_BOVIN

Integrin alpha-V

ITGAV

Biological adhesion

 

 F1MK44_BOVIN

Integrin alpha-5

ITGA5

  

 TTHY_BOVIN

Transthyretin

TTR

localization

Transporter activity

 F1MC45_BOVIN

Complement factor H (Fragment)

CFH

  

 J9QD97_BOVIN

Periostin variant 9

   

 ACTS_BOVIN

Actin, alpha skeletal muscle

ACTA1

Cellular component organization or biogenesis/Cellular process/Developmental process/localization

Structural molecule activity

 E1B9K1_BOVIN

Polyubiquitin-C

UBC

  

 A7YWR0_BOVIN

Apolipoprotein E

APOE

  

 FA9_BOVIN

Coagulation factor IX

F9

Apoptotic process/Biological regulation/Developmental process/Immune system process/ localization/Metabolic process/Multicellular organismal process/Response to stimulus

Binding/Catalytic activity/Enzyme regulator activity/Receptor activity

 COMP_BOVIN

Cartilage oligomeric matrix protein

COMP

  

 K2C80_BOVIN

Keratin, type II cytoskeletal 80

KRT80

Cellular component organization or biogenesis/Cellular process/Developmental process

Structural molecule activity

 TRFE_BOVIN

Serotransferrin

TF

localization/Metabolic process

Catalytic activity

 K4JDR8_BOVIN

Alpha-2-macroglobulin variant 5

A2M

  

 Q32P72_BOVIN

CP protein (Fragment)

CP

  

 J9ZW47_BOVIN

Integrin beta

   

 F1MM86_BOVIN

Complement component C6

C6

  

 E1BI02_BOVIN

Fibromodulin

FMOD

  

 VNN1_BOVIN

Pantetheinase

VNN1

Biological adhesion/Cellular process/Metabolic process

Catalytic activity

 G3X807_BOVIN

Histone H4 (Fragment)

 

Cellular component organization or biogenesis/Cellular process/Metabolic process

Binding

 MOT1_BOVIN

Monocarboxylate transporter 1

SLC16A1

Cellular process/localization

Transporter activity

 TF_BOVIN

Tissue factor

F3

Biological regulation/Cellular process/Response to stimulus

Binding/Receptor activity

 HS71L_BOVIN

Heat shock 70 kDa protein 1-like

HSPA1L

Metabolic process/Response to stimulus

 

 Q3ZCA7_BOVIN

Guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3

GNAI3

Biological regulation/Cellular process/Metabolic process/Response to stimulus

Binding/Catalytic activity

 IDHC_BOVIN

Isocitrate dehydrogenase [NADP] cytoplasmic

IDH1

  

 Q1PBC8_BOVIN

CD14 (Fragment)

   

 F1MJJ8_BOVIN

Radixin (Fragment)

RDX

  

 IF4A2_BOVIN

Eukaryotic initiation factor 4A-II

EIF4A2

Biological regulation/Metabolic process

Binding/Catalytic activity/Translation regulator activity

 C1QB_BOVIN

Complement C1q subcomponent subunit B

C1QB

  

 A6QPD4_BOVIN

LOC790886 protein

LOC790886

  

 CTL2_BOVIN

Choline transporter-like protein 2

SLC44A2

localization

Transporter activity

 HPCL1_BOVIN

Hippocalcin-like protein 1

HPCAL1

Cellular process/Multicellularorganismal process

 

 Q24K07_BOVIN

Vacuolar protein sorting 11 homolog (S. cerevisiae)

VPS11

  

 Q5H9M6_BOVIN

Dynein heavy chain (Fragment)

Bv2

  

 Q864S1_BOVIN

Cathepsin C (Fragment)

   

 Q4ZJS0_BOVIN

MHC class I antigen (Fragment)

BoLA-N

  

 Q58CZ4_BOVIN

Flotillin 2

FLOT2

  

 MBL2_BOVIN

Mannose-binding protein C

MBL

 

Binding

 TM214_BOVIN

Transmembrane protein 214

TMEM214

  

 Q8MIR1_BOVIN

Nicotinic acetylcholine receptor beta 2 subunit (Fragment)

CHRNB2

  

 Q5E9W1_BOVIN

CDC45-like

CDC45L

  

Mass spectrometric (with a set FDR of 5 %) identification of proteins was present in exosomes generated by ICAR cultured at 1 % O2 and at 8 % O2. Data were subjected to ontology and pathway analysis using PANTHER and gene ontology algorithms and classified based on biological process and molecular function

Fig. 5

Proteomic analysis of bovine endometrial ICAR-derived exosomes. Mass spectrometric analyses of ICAR cell-derived exosome proteins. a Representative Venn diagram of common and unique proteins identified by 5600 Triple TOF MS (ABSciex) from exosomes released by ICAR cells at 48 h at both 8 % O2 and 1 % O2. b The gene ontology classification of ICAR cell-derived exosome proteins, on the basis of their involvement in biological process, identified clusters that are unique to and present only in exosomes of ICAR cultured at 1 % O2 but not those at 8 % O2. These biological processes were: growth (0.7 %), locomotion (0.7 %) and reproduction (1.4 %). c Molecular function (using PANTHER and Gene Ontology algoritnms) of exosome proteins were mostly related to binding and catalytic activity in both ICAR cultured at 1 % O2 and at 8 % O2

Discussion

A successful pregnancy is dependent of having a quality embryo and a receptive uterus synergizing with a synchronized crosstalk between the endometrium and embryo. Any insults or disturbances to its normal course can compromise implantation and the ability for the growing fetus to develop properly in the uterus [26]. The endometrium clearly has important functions in dairy cow pregnancy and we have now shown that exosomal release (30–120 nm) is part of its armamentarium which has analogous properties to similar tissues of other mammalian species.

In the present case, we have shown for the first time the effects of hypoxia on the biological activities of endometrial ICAR cells, including actions on the release and protein content of exosomes. Although it remains to be determined whether exosomes released from ICAR cells at different oxygen tensions also serve different functional goals, our data underscore that the content of exosomes may reflect the physiological state of the cells.

Our non-exosomal characterization of the ICAR cells indicated that the migration and proliferative capacity of ICAR cells decreased, while activation of apoptotic caspase-3 was enhanced at 1 % O2 (hypoxia), compared with an oxygen tension that was close to the bovine endometrial physiological oxygen levels (8 % O2; [38]). Moreover, the effect on migration was greater when exposed at 1 % O2 [39]. Interestingly, no relationship between oxygen tension and cell proliferation and apoptosis was observed in this previous study. Differences in cell types may explain this observation. Ito et al. described the rate of proliferation of human mesenchymal stem cell (MSCs) was observed to be highest in 5 % O2 and the lowest in < 0.1 % O2 conditions [40]. The MSCs at severely induced hypoxic conditions (<0.1 % O2), showed a decrease in proliferative ability, but were able to maintain viability for at least 48 h through increased glucose availability, to facilitate the generation of energy. Similar results were obtained from an airway smooth muscle study [41]. Hence, our cells have relatively normal proliferation responses to decreased oxygen tension.

Our study suggests that exosomes can serve as a vector for signaling molecules that harbor a variety of bioactive molecules including proteins at the conceptus-endometrial interface and that has the potential to modulate the functions of targeted cells during early pregnancy. Endometrial exosome release may also be modulated during an insult such as infection [42, 43]. In the current study we utilized hypoxia (i.e. 1 % O2) as a known modulator of exosome release as documented by alteration to both the number of exosomes released as well as differences in the exosomal content (cargo) [24, 27, 29].

In our study, endometrial cells exposed to 1 % O2released ~3.6 more exosomes relative to the 8 % O2 culture treatment, suggesting that hypoxia modulates cell function, including the release of exosomes. Hypoxia has already been reported to be a stimulus to increase secretion of exosomes by several groups [4446]. It is also suggested that the protein and RNA content of exosomes can reflect the physiological state of the cell as well as when the cells are in stress condition [47, 48]. However, the initial stress insult that contributed to an alteration of the exosomal content in relation to the functional effects of the subsequent cargo transfer and their role in cell-to-cell communication remains unclear. It is possible that exposure to other stressors such as adverse environmental hazards [4951] will also increase secretion of exosomes and alter composition of the cargo.

The protein content of exosomes from ICAR cells cultured under the 1 % O2 contained unique proteins compared to the contents of the ICAR exosomes cultured at 8 % O2. Our proteomic analyses detected the presence of tetraspanin-6 (TSPAN6), disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) that are only unique to exosomes of ICAR cultured at 1 % O2. These proteins are involved in the biological processes for reproduction. Interestingly, to evaluate TSPAN6, belonging to the transmembrane 4 superfamily that mediate the regulation of signal transduction events, as well as the disintegrin-like metalloproteinase ADAM10 which participates in ectodomain shedding activity could provide great insights into their functional role and regulation that is important for reproduction.

Studies using immunohistochemistry of human placental explants [52] have demonstrated that ADAM10 expression is significantly increased in preeclamptic placentas compared with normal placentas. Up-regulation of ADAM10 could induce placental release of soluble vascular endothelial growth factor receptor-1 (sFlt-1) and this cascade is associated with endothelial dysfunction, suggesting the significant role of oxidative change in preeclamptic placentas. ADAM10 is also a sheddase [53] that could induce CD46 shedding attributed to cell apoptotic processes [54], as well as mediate E-cadherin shedding affecting cellular adhesion and cell migration [55].

Mass spectrometry detection of pantetheinase (VNN1) in exosomes was unique to ICAR cultured at 8 % O2. VNN1 is an enzyme that hydrolyses pantetheine to form pantothenic acid (a precursor of coenzyme A) and the antioxidant cysteamine [56]. VNN1 could promote tissue inflammation through peroxisome proliferator-activated receptor gamma as well as modulate levels of glutathione [57]. It is proposed that VNN1 have innate immune functions and might contribute to tissue injury in endometritis [58, 59]. VNN1 was also reported being involved in proteolysis and can denature proteins by reducing disulfides [60], suggesting that it may have a role in regulating uterine receptivity for implantation and trophoblast invasion [61].

Mass spectrometry detected kininogen-2 (KNG2) in exosomes generated by ICAR cells cultured at either 1 or 8 % O2. KNG2 is a precursor protein to high molecular weight kininogen, low molecular weight kininogen and bradykinin and the concentration were reported to fluctuate during ovulation, pregnancy, and parturition [62]. Studies also showed that the release of vasoactive bradykinins from high molecular weight kininogen and low molecular weight kininogen are responsible for micro-vascular permeability and vascular growth, which plays an essential role in utero-placental vasculature and angiogenesis, necessary for embryonic and fetal survival [63].

Conclusion

Our present findings show that ICAR cell function, release of exosomes and exosomal content can be altered when subjected to adverse stimuli. These findings should be expanded to include cells of endometrial epithelial origin, interactions between these cells (i.e. stromal—epithelial crosstalk) and in the presence of common pathophysiological factors associated with reduced fertility (e.g. infectious or inflammatory agents). The identification of unique proteins (by mass spectrometry) in exosomes of ICAR cultured at 1 % O2 compared to 8 % O2 suggests that the cells respond and release proteins encapsulated within the exosomes to signal the environment in which they live. It is hoped that identification of unique proteins in exosomes following stimulation by factors affecting the physiological condition of cows may lead to novel targets for manipulation to aid fertility. Moreover, investigations into the release, uptake and content of exosomes may offer the opportunity to evaluate maternal-fetal crosstalk.

Abbreviations

ADAM 10: 

Metalloproteinase domain-containing protein 10

DTT: 

Dithiothreitol

FDR: 

False discovery rate

ICAR: 

Intercaruncular stromal cell

KNG2: 

Kininogen-2

LDH: 

Lactate dehydrogenase

MS/MS: 

Mass spectrometry/mass spectrometry

PANTHER: 

Protein analysis through evolutionary relationships

PBS: 

Phosphate buffered saline

PGF

Prostaglandin F

PVDF: 

Polyvinylidene fluoride

RIPA: 

Radioimmunoprecipitation assay buffer

RWD: 

Relative wound density

sFLT-1: 

Soluble vascular endothelial growth factor receptor-1

TSPAN6: 

Tetraspanin-6

VNN1: 

Pantetheinase

Declarations

Acknowledgments

The authors acknowledge the assistance of Dr. Jamie Riches and Dr. Rachel Hancock of the Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology (QUT) for the electron microscope analyses.

We also thank our colleagues at DairyNZ for their helpful insights. YQ Koh is supported by a student scholarship from a partnership fund (DRCX1302) between the New Zealand Ministry of Business, Innovation and Employment and New Zealand dairy farmers through DairyNZ Inc. CS holds a research fellowship at The University of Queensland Centre for Clinical Research, Brisbane, Australia. GER was in receipt of an NMHRC Principal Research Fellowship. These studies were funded in part by the Australian Research Council, Therapeutic Innovation Australian and National Collaborative Research Infrastructure Strategy.

Funding

Australian Research Council and a partnership fund (DRCX1302) between the New Zealand Ministry of Business, Innovation and Employment and New Zealand dairy farmers through DairyNZ Inc.

Availability of data and materials

The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Authors’ contributions

YQK performed the study, collected and interpreted data performing statistical analysis and wrote the manuscript. YQK, SR, HNP and KV performed mass spectrometry analyses and reviewed the data generated. YQK, CS, HNP, GER and MDM were responsible for the study concept and participated in designing the study and interpreted data. CS, GER, HNP, MDM revised and approved the final version of manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
University of Queensland Centre for Clinical Research, The University of Queensland

References

  1. Banu SK, Arosh JA, Chapdelaine P, Fortier MA. Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy. Biol Reprod. 2005;73:230–6.View ArticlePubMedGoogle Scholar
  2. Verduzco A, Fecteau G, Lefebvre R, Smith LC, Murphy BD. Expression of steroidogenic proteins in bovine placenta during the first half of gestation. Reprod Fertil Dev. 2012;24:392–404.View ArticlePubMedGoogle Scholar
  3. Mansouri-Attia N, Aubert J, Reinaud P, Giraud-Delville C, Taghouti G, Galio L, Everts RE, Degrelle S, Richard C, Hue I, et al. Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation. Physiol Genomics. 2009;39:14–27.View ArticlePubMedGoogle Scholar
  4. Arosh JA, Banu SK, Chapdelaine P, Fortier MA. Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy. Endocrinology. 2004;145:407–17.View ArticlePubMedGoogle Scholar
  5. Gray CA, Bartol FF, Tarleton BJ, Wiley AA, Johnson GA, Bazer FW, Spencer TE. Developmental biology of uterine glands. Biol Reprod. 2001;65:1311–23.View ArticlePubMedGoogle Scholar
  6. Ashley RL, Antoniazzi AQ, Anthony RV, Hansen TR. The chemokine receptor CXCR4 and its ligand CXCL12 are activated during implantation and placentation in sheep. Reprod Biol Endocrinol. 2011;9:148.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Bauersachs S, Wolf E. Immune aspects of embryo-maternal cross-talk in the bovine uterus. J Reprod Immunol. 2013;97:20–6.View ArticlePubMedGoogle Scholar
  8. Asselin E, Drolet P, Fortier MA. In vitro response to oxytocin and interferon-Tau in bovine endometrial cells from caruncular and inter-caruncular areas. Biol Reprod. 1998;59:241–7.View ArticlePubMedGoogle Scholar
  9. Turner ML, Cronin JG, Healey GD, Sheldon IM. Epithelial and stromal cells of bovine endometrium have roles in innate immunity and initiate inflammatory responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2, TLR1, and TLR6. Endocrinology. 2014;155:1453–65.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Arnold JT, Kaufman DG, Seppala M, Lessey BA. Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model. Hum Reprod. 2001;16:836–45.View ArticlePubMedGoogle Scholar
  11. Okuda K, Kasahara Y, Murakami S, Takahashi H, Woclawek-Potocka I, Skarzynski DJ. Interferon-tau blocks the stimulatory effect of tumor necrosis factor-alpha on prostaglandin F2alpha synthesis by bovine endometrial stromal cells. Biol Reprod. 2004;70:191–7.View ArticlePubMedGoogle Scholar
  12. Asselin E, Goff AK, Bergeron H, Fortier MA. Influence of sex steroids on the production of prostaglandins F2 alpha and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol Reprod. 1996;54:371–9.View ArticlePubMedGoogle Scholar
  13. Krishnaswamy N, Chapdelaine P, Tremblay JP, Fortier MA. Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line. Endocrinology. 2009;150:485–91.View ArticlePubMedGoogle Scholar
  14. Ng YH, Rome S, Jalabert A, Forterre A, Singh H, Hincks CL, Salamonsen LA. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One. 2013;8:e58502.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Ruiz-Gonzalez I, Xu J, Wang X, Burghardt RC, Dunlap KA, Bazer FW. Exosomes, endogenous retroviruses and toll-like receptors: pregnancy recognition in ewes. Reproduction. 2015;149:281–91.View ArticlePubMedGoogle Scholar
  16. Cleys ER, Halleran JL, McWhorter E, Hergenreder J, Enriquez VA, da Silveira JC, Bruemmer JE, Winger QA, Bouma GJ. Identification of microRNAs in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep. Mol Reprod Dev. 2014;81:983–93.View ArticlePubMedGoogle Scholar
  17. Burns G, Brooks K, Wildung M, Navakanitworakul R, Christenson LK, Spencer TE. Extracellular vesicles in luminal fluid of the ovine uterus. PLoS One. 2014;9:e90913.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Sohel MM, Hoelker M, Noferesti SS, Salilew-Wondim D, Tholen E, Looft C, Rings F, Uddin MJ, Spencer TE, Schellander K, Tesfaye D. Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: implications for Bovine Oocyte Developmental Competence. PLoS One. 2013;8:e78505.View ArticlePubMedPubMed CentralGoogle Scholar
  19. van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012;64:676–705.View ArticlePubMedGoogle Scholar
  20. Simons M, Raposo G. Exosomes--vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009;21:575–81.View ArticlePubMedGoogle Scholar
  21. Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway. Am J Physiol Heart Circ Physiol. 2007;292:H3052–3056.View ArticlePubMedGoogle Scholar
  22. Orriss IR, Knight GE, Utting JC, Taylor SE, Burnstock G, Arnett TR. Hypoxia stimulates vesicular ATP release from rat osteoblasts. J Cell Physiol. 2009;220:155–62.View ArticlePubMedGoogle Scholar
  23. Wysoczynski M, Ratajczak MZ. Lung cancer secreted microvesicles: underappreciated modulators of microenvironment in expanding tumors. Int J Cancer. 2009;125:1595–603.View ArticlePubMedPubMed CentralGoogle Scholar
  24. King HW, Michael MZ, Gleadle JM. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer. 2012;12:421.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Maltepe E, Saugstad OD. Oxygen in health and disease: regulation of oxygen homeostasis--clinical implications. Pediatr Res. 2009;65:261–8.View ArticlePubMedGoogle Scholar
  26. Mallard EC, Rees S, Stringer M, Cock ML, Harding R. Effects of chronic placental insufficiency on brain development in fetal sheep. Pediatr Res. 1998;43:262–70.View ArticlePubMedGoogle Scholar
  27. Park JE, Tan HS, Datta A, Lai RC, Zhang H, Meng W, Lim SK, Sze SK. Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Mol Cell Proteomics. 2010;9:1085–99.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Salomon C, Kobayashi M, Ashman K, Sobrevia L, Mitchell MD, Rice GE. Hypoxia-induced changes in the bioactivity of cytotrophoblast-derived exosomes. PLoS One. 2013;8:e79636.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Onogi A, Naruse K, Sado T, Tsunemi T, Shigetomi H, Noguchi T, Yamada Y, Akasaki M, Oi H, Kobayashi H. Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy. Placenta. 2011;32:665–70.View ArticlePubMedGoogle Scholar
  30. Fortier MA, Guilbault LA, Grasso F. Specific properties of epithelial and stromal cells from the endometrium of cows. J Reprod Fertil. 1988;83:239–48.View ArticlePubMedGoogle Scholar
  31. Salomon C, Ryan J, Sobrevia L, Kobayashi M, Ashman K, Mitchell M, Rice GE. Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis. PLoS One. 2013;8:e68451.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Kobayashi M, Salomon C, Tapia J, Illanes SE, Mitchell MD, Rice GE. Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200. J Transl Med. 2014;12:4.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Salomon C, Torres MJ, Kobayashi M, Scholz-Romero K, Sobrevia L, Dobierzewska A, Illanes SE, Mitchell MD, Rice GE. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration. PLos One. 2014;9(6):e98667.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Brinkman DL, Jia X, Potriquet J, Kumar D, Dash D, Kvaskoff D, Mulvenna J. Transcriptome and venom proteome of the box jellyfish Chironex fleckeri. BMC Genomics. 2015;16:407.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Vaswani K, Ashman K, Reed S, Salomon C, Sarker S, Arraztoa JA, Perez-Sepulveda A, Illanes SE, Kvaskoff D, Mitchell MD, Rice GE. Applying SWATH mass spectrometry to investigate human cervicovaginal fluid during the menstrual cycle. Biol Reprod. 2015;93:39.View ArticlePubMedGoogle Scholar
  36. Rappsilber J, Ishihama Y, Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem. 2003;75:663–70.View ArticlePubMedGoogle Scholar
  37. Thomas PD, Campbell MJ, Kejariwal A, Mi H, Karlak B, Daverman R, Diemer K, Muruganujan A, Narechania A. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 2003;13:2129–41.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Gahlenbeck H, Frerking H, Rathschlag-Schaefer AM, Bartels H. Oxygen and carbon dioxide exchange across the cow placenta during the second part of pregnancy. Respir Physiol. 1968;4:119–31.View ArticlePubMedGoogle Scholar
  39. Ng CT, Biniecka M, Kennedy A, McCormick J, Fitzgerald O, Bresnihan B, Buggy D, Taylor CT, O’Sullivan J, Fearon U, Veale DJ. Synovial tissue hypoxia and inflammation in vivo. Ann Rheum Dis. 2010;69:1389–95.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Ito A, Aoyama T, Yoshizawa M, Nagai M, Tajino J, Yamaguchi S, Iijima H, Zhang X, Kuroki H. The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16(INK4A) mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent. J Stem Cells Regen Med. 2015;11:25–31.PubMedPubMed CentralGoogle Scholar
  41. Cogo A, Napolitano G, Michoud MC, Barbon DR, Ward M, Martin JG. Effects of hypoxia on rat airway smooth muscle cell proliferation. J Appl Physiol (1985). 2003;94:1403–9.View ArticleGoogle Scholar
  42. Harp D, Driss A, Mehrabi S, Chowdhury I, Xu W, Liu D, Garcia-Barrio M, Taylor RN, Gold B, Jefferson S, et al. Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro. Cell Tissue Res. 2016;365:187–96.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Nudel K, Massari P, Genco CA. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2. Infect Immun. 2015;83:3410–7.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Lee SM, Romero R, Lee YJ, Park IS, Park CW, Yoon BH. Systemic inflammatory stimulation by microparticles derived from hypoxic trophoblast as a model for inflammatory response in preeclampsia. Am J Obstet Gynecol. 2012;207(4):337.e1–8.View ArticleGoogle Scholar
  45. Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringner M, Morgelin M, Bourseau-Guilmain E, Bengzon J, Belting M. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci U S A. 2013;110:7312–7.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Sano S, Izumi Y, Yamaguchi T, Yamazaki T, Tanaka M, Shiota M, Osada-Oka M, Nakamura Y, Wei M, Wanibuchi H, et al. Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3 T3-L1 cells. Biochem Biophys Res Commun. 2014;445:327–33.View ArticlePubMedGoogle Scholar
  47. Belting M, Christianson HC. Role of exosomes and microvesicles in hypoxia-associated tumour development and cardiovascular disease. J Intern Med. 2015;278:251–63.View ArticlePubMedGoogle Scholar
  48. de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, Schiffelers RM, Gucek M, van Balkom BW: Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell Vesicles. 2012;1. http://www.journalofextracellularvesicles.net/index.php/jev/article/view/18396.
  49. Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, Wood MJ, Cooper JM. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol Dis. 2011;42:360–7.View ArticlePubMedPubMed CentralGoogle Scholar
  50. Fevrier B, Vilette D, Archer F, Loew D, Faigle W, Vidal M, Laude H, Raposo G. Cells release prions in association with exosomes. Proc Natl Acad Sci U S A. 2004;101:9683–8.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Pan-Montojo F, Reichmann H. Considerations on the role of environmental toxins in idiopathic Parkinson’s disease pathophysiology. Transl Neurodegener. 2014;3:10.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Zhao S, Gu Y, Fan R, Groome LJ, Cooper D, Wang Y. Proteases and sFlt-1 release in the human placenta. Placenta. 2010;31:512–8.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Bouillot S, Tillet E, Carmona G, Prandini MH, Gauchez AS, Hoffmann P, Alfaidy N, Cand F, Huber P. Protocadherin-12 cleavage is a regulated process mediated by ADAM10 protein: evidence of shedding up-regulation in pre-eclampsia. J Biol Chem. 2011;286:15195–204.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Hakulinen J, Keski-Oja J. ADAM10-mediated release of complement membrane cofactor protein during apoptosis of epithelial cells. J Biol Chem. 2006;281:21369–76.View ArticlePubMedGoogle Scholar
  55. Maretzky T, Reiss K, Ludwig A, Buchholz J, Scholz F, Proksch E, de Strooper B, Hartmann D, Saftig P. ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation. Proc Natl Acad Sci U S A. 2005;102:9182–7.View ArticlePubMedPubMed CentralGoogle Scholar
  56. Martin F, Malergue F, Pitari G, Philippe JM, Philips S, Chabret C, Granjeaud S, Mattei MG, Mungall AJ, Naquet P, Galland F. Vanin genes are clustered (human 6q22-24 and mouse 10A2B1) and encode isoforms of pantetheinase ectoenzymes. Immunogenetics. 2001;53:296–306.View ArticlePubMedGoogle Scholar
  57. Dammanahalli KJ, Stevens S, Terkeltaub R. Vanin-1 pantetheinase drives smooth muscle cell activation in post-arterial injury neointimal hyperplasia. PLoS One. 2012;7:e39106.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Hayes MA, Quinn BA, Lillie BN, Cote O. Changes in various endometrial proteins during cloprostenol-induced failure of early pregnancy in mares. Anim Reprod. 2012;9:723–41.Google Scholar
  59. Berruyer C, Martin FM, Castellano R, Macone A, Malergue F, Garrido-Urbani S, Millet V, Imbert J, Dupre S, Pitari G, et al. Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress. Mol Cell Biol. 2004;24:7214–24.View ArticlePubMedPubMed CentralGoogle Scholar
  60. Kaskow BJ, Proffitt JM, Blangero J, Moses EK, Abraham LJ. Diverse biological activities of the vascular non-inflammatory molecules - the Vanin pantetheinases. Biochem Biophys Res Commun. 2012;417:653–8.View ArticlePubMedGoogle Scholar
  61. Mullen MP, Elia G, Hilliard M, Parr MH, Diskin MG, Evans AC, Crowe MA. Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle. J Proteome Res. 2012;11:3004–18.View ArticlePubMedGoogle Scholar
  62. Karkkainen T, Hamberg U. Kininogen as a pregnancy-associated plasma protein. Adv Exp Med Biol. 1986;198(Pt A):167–72.View ArticlePubMedGoogle Scholar
  63. Vonnahme KA, Fernando SC, Ross JW, Ashworth MD, DeSilva U, Malayer JR, Geisert RD. Porcine endometrial expression of kininogen, factor XII, and plasma kallikrein in cyclic and pregnant gilts. Biol Reprod. 2004;70:132–8.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2016

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