Characterization of exosomal release in bovine endometrial intercaruncular stromal cells

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.


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 2α ) 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 cytokeratinnegative 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 [15][16][17][18] 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 [21][22][23][24]. 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.

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 cm 2 (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 % CO 2 -balanced N 2 to obtain a hypoxic (1 % O 2 ) environment or under physiologically relevant conditions (8 % O 2 ). 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 % O 2 and 1 % O 2 and ICAR cell viability was accessed. No significant difference in the LDH activity was observed (data not shown) between 8 % O 2 and 1 % O 2 , 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 % O 2 or 8 % O 2 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 % O 2 or 8 % O 2 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 % O 2 or 8 % O 2 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, Swingingbucket 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.

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 reequilibrated with 5 % Buffer B for 6 min. The in-depth proteomic analysis was performed using the Information Dependent Acquisition (IDA) experiments on the Triple-TOF® 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 Pro-teinPilot™ 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 % O 2 ) with the control group (i.e. 8 % O 2 ) using a commercially-available software package (Prism 6, GraphPad Inc, La Jolla, CA 92037 USA).

The Effect of Oxygen Tension on Bovine Endometrial (ICAR) cell migration and proliferation
The effect of normal oxygen tension (i.e. 8 % O 2 ) and hypoxia (i.e. 1 % O 2 ) 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 % O 2 (2173 ± 36 and 2620 ± 50 for 1 % O 2 and 8 % O 2 , 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 % O 2 , the proliferative capacity of ICAR cells was inhibited (p < 0.05)~32 % compared with 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).

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 % O 2 and 1 % O 2 , respectively) ( Fig. 4c). Interestingly,

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 % O 2 and at 8 % O 2 128 proteins identified as unique to exosomes of ICAR cultured at 1 % O 2 ; 46 proteins were identified as unique to exosomes of ICAR cultured at 8 % O 2 (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 % O 2 but not those at 8 % O 2 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 % O 2 and to those of ICAR cultured at 8 % O 2 (Fig. 5c).

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 % O 2 (hypoxia), compared with an oxygen tension that was close to the bovine           [38]). Moreover, the effect on migration was greater when exposed at 1 % O 2 [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.

CDC45-like CDC45L
Mass spectrometric (with a set FDR of 5 %) identification of proteins was present in exosomes generated by ICAR cultured at 1 % O 2 and at 8 % O 2 . Data were subjected to ontology and pathway analysis using PANTHER and gene ontology algorithms and classified based on biological process and molecular function described the rate of proliferation of human mesenchymal stem cell (MSCs) was observed to be highest in 5 % O 2 and the lowest in < 0.1 % O 2 conditions [40]. The MSCs at severely induced hypoxic conditions (<0.1 % O 2 ), 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 conceptusendometrial 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 % O 2 ) 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 % O 2 -released~3.6 more exosomes relative to the 8 % O 2 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 [44][45][46]. 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 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 % O 2 but not those at 8 % O 2 . 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 % O 2 and at 8 % O 2 cell-to-cell communication remains unclear. It is possible that exposure to other stressors such as adverse environmental hazards [49][50][51] will also increase secretion of exosomes and alter composition of the cargo.
The protein content of exosomes from ICAR cells cultured under the 1 % O 2 contained unique proteins compared to the contents of the ICAR exosomes cultured at 8 % O 2 . 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 % O 2 . 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 Ecadherin shedding affecting cellular adhesion and cell migration [55].
Mass spectrometry detection of pantetheinase (VNN1) in exosomes was unique to ICAR cultured at 8 % O 2 . 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 % O 2 . 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 % O 2 compared to 8 % O 2 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.