Carmen Alminana-Brines

Emilie Corbin
email: emilie.corbin@inra.fr

Guillaume Tsikis
email: guillaume.tsikis@inra.fr

Agostinho Soares

Alcantara Neto

Valérie Labas
email: valerie.labas@inra.fr

Karine Reynaud
email: kreynaud@vet-alfort.fr

Laurent Galio
email: laurent.galio@inra.fr

Rustem Uzbekov
email: rustem.uzbekov@univ-tours.fr

Anastasiia S Garanina
email: anastasiacit@gmail.com

Xavier Druart
email: xavier.druart@inra.fr

Carmen Almiñana
email: calminana@inra.fr

Agostinho S Alcântara-Neto
email: agostinho.alcantara@inra.fr

Pascal Mermillod
email: pascal.mermillod@inra.fr

Leese Hj

Hugentobler Sa

Gray Sm

Morris Dg

Sturmey Rg

Reprod Fertil Dev

R Lopera-Vasquez

Gutierrez-Adan A Yanez

Gutierrez-Adan A Bermejo-Alvarez

Ramirez Ma

Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk

Keywords: Extracellular vesicles, Oviduct, Embryo development, Embryomaternal interactions, proteomic

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (p-value<0.01). Interestingly, 97 were exclusively expressed in in vivo EVs, 47 were present only in in vitro and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in in vitro EVs. Moreover, we showed that in vitro produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. In vivo EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under in vitro conditions.

INTRODUCTION

Successful pregnancy requires an appropriate communication between the female reproductive tract and the embryo(s). Disturbance in this unique communication system is associated with high rates of early pregnancy loss and it is becoming increasingly evident that it also influences the developmental potential of the offspring into adulthood (Baker 1998, Mahsoudi et al. 2007). Strong evidence exists with respect to the signals exchange between the early embryo and the oviduct leading to an appropriate embryo development and successful pregnancy (Lee et al. 2002[START_REF] Alminana | Early developing pig embryos mediate their own environment in the maternal tract[END_REF], Maillo et al. 2015). Absence of these oviduct signals in ART have raised the question of how much these techniques can affect the outcomes (Ostrup et al. 2011, O'Neill et al. 2012). Significant gains in our understanding of the essential embryotrophic components of the oviduct fluid and their interactions with the embryo have been achieved in the last years (Georgiou et al. 2007, Leese et al. 2008, Aviles et al. 2010, Schmaltz-Panneau et al. 2014). However, there is a need for further exploring the contribution of the oviduct to the reproductive success.

Recently, exosomes and microvesicles, have been identified as essential components of uterine (Ng et al. 2013, Burns et al. 2014) and oviduct fluids [START_REF] Strehler | Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm[END_REF], Lopera-Vasquez et al. 2017). Exosomes are 30-150 nm vesicles of endocytotic origin released upon fusion of a multi-vesicular body with the cell membrane, while microvesicles are 100-1000 nm in diameter and bud directly from the cell membrane (Colombo et al. 2014). Both are collectively known as extracellular vesicles (EVs), and are considered important tools in cell-to-cell communication (Valadi et al. 2007) by transferring their molecular cargo (proteins, mRNA, miRNA) from one cell to another.

In the maternal tract, they have been proposed as important tools to regulate Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 5 5 gamete/embryo-maternal interactions [START_REF] Martin-Deleon | Role of exosomes in the reproductive tract Oviductosomes mediate interactions of oviductal secretion with gametes/early embryo[END_REF], Burns et al. 2016). However, while different studies have evaluated the EVs secretion/content (proteins, miRNA) produced by the uterus from in vivo (Ng et al. 2013, Burns et al. 2016) and in vitro origin (Greening et al. 2016, Bidarimath et al. 2017), none have provided an extensive characterization of oviduct EVs content up to date. An important requisite to decipher the possible role of the EVS in the embryo-oviduct dialogue. Only one protein, PMCA4a, which is essential for sperm hyperactivated motility and fertility have been identified in oviduct EVs [START_REF] Strehler | Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm[END_REF]. Despite our lack of knowledge about their content, the EVs derived from in vivo oviduct fluid and in vitro culture of bovine oviduct epithelial cells (BOEC), seem to improve the cryotolerance of in vitro produced embryos (Lopera-Vasquez et al. 2016, Lopera-Vasquez et al. 2017).

Given the known differences in secretions by BOEC in vivo and in vitro (Rottmayer et al. 2006), and the increasing number of studies based on EVs derived from in vitro primary cell culture or cell lines, a comparative study of the EVs content of in vivo and in vitro origin seems imperative. Thus, we aimed at: 1) deciphering the oviduct EVs protein content from in vivo and in vitro origin; 2) analyzing whether embryos are able to internalize oviduct EVs and 3) investigating their functional effect on embryo development. For this purpose, a bovine model was used, since bovine has been demonstrated to be a valuable experimental model for addressing ART-related questions.

MATERIAL AND METHODS

Collection of bovine oviduct fluid and epithelial cells (BOEC)

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 6 6

Oviducts and ovaries were obtained from cows at local slaughterhouse (Sablé sur Sarthe, France), with the permission of the direction of the slaughterhouse and the agreement of local sanitary services. Oviducts and their attached ovaries were transported to the laboratory at 37ºC within 2-3 h after collection. For all experiments, ipsilateral and contralateral oviducts from the same animal at the post-ovulatory stage of the bovine estrous cycle were used. Animals showing recent ovulation sites in the attached ovaries, indicating they were at postovulatory stage (1-5 days of estrus cycle),

were selected for EVs collection. To minimize variability, the same oviducts were used for in vivo EVs collection by oviduct flushing than for in vitro EVs collection, by using the conditioned media after in vitro BOEC primary culture. First, oviducts were dissected free from surrounding tissues. Subsequently, to recover the oviduct flushing the lumen of the oviduct was flushed with 500µl of sterile PBS (Sigma P4417-TAB, Lyon, France). Then, BOEC were isolated by mechanical scraping of the oviduct with a slide for primary in vitro BOEC culture as described by Van Langendonckt and colleagues (Van Langendonckt et al. 1995). BOEC were washed three times by sedimentation in 10mL of Tissue culture medium-199-Hepes (TCM-199, Sigma M7528) supplemented with bovine serum albumin (BSA stock fraction V, Sigma A9647) and 8µl/mL gentamycin (Sigma G1272). The resulting cellular pellet was diluted 100 times in culture medium consisting in supplemented with 10% heat-treated foetal calf serum (FCS, Sigma F9665) and 8µl/mL gentamycin before seeding. At this point, an aliquot of in vivo BOEC (from the day (day 0) of collection) was stored at -20ºC for further comparative protein analysis with in vitro BOEC and EVs by Western blotting, while the rest of BOEC were seeded for culture.

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk.

Isolation of EVs from in vivo and in vitro origin

Oviduct flushings from different animals were pooled (n=3animals per replicate; in 4 replicates). Conditioned media obtained from different 25cm2 flasks were also pooled (total of 100 mL/replicate; in 4 replicates). EVs were obtained from oviduct flushings and conditioned media by serial centrifugation as described by Théry et al., (Thery et al. 2006). First, flushing and conditioned media were centrifuged at 300 g for 15 min, followed by 12,000 g for 15 min to remove cells, blood, and cell debris and ultracentrifuged twice at 100,000 g for 90 min (BECKMAN L8-M; SW41T1 rotor) to

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 8 8 pellet exosomes. The pellets were resuspended in 100 µL of PBS and stored at -20ºC for further analysis.

Transmission electron microscopy (TEM)

Vesicle suspensions were diluted in PBS to attain a protein concentration of 0.6 µg per µL. Then, 3µl of the sample were placed on the formvar carbon-coated grid for 5 min and washed with distilled water (three times). For negative contrast the samples were stained with 2% uranyl acetate for 2 min and left to dry. The micrographs were obtained using TEM HITACHI HT 7700 Elexience at 80kV (with a charge-coupled device camera AMT) and JEM 1011 (JEOL, Japan) equipped with a Gatan digital camera driven by Digital Micrograph software (Gatan, Pleasanton, USA) at 100 kV. The processing of the photos and exosome size calculation were carried out by ImageJ software.

EVs labeling, EVs-embryo co-incubation and observation

In vivo EVs preparations from oviduct flushings (pool of three animals; 3 replicates) were labeled with a lipophilic green fluorescence dye (PKH67, Sigma, France) as described by Saadeldin, et al., (Saadeldin et al. 2014). PKH67 is a widely-used dye for visualization of exosomes uptake by cells (Burns et al. 2014, Saadeldin et al. 2014).

First, a dilution of the EVs preparation was performed by mixing 25µL of the EVs suspension in PBS with 125µL of diluent C (Cell mixture). In addition, 25µL of PBS were mixed with 125µL of diluent C as a negative control. Then, the dye dilution was prepared by adding 1µL dye to 250µL of diluent C and 125µL of this mixture were added to EVs and Control mixtures and incubated for 5 min at room temperature (Final Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 9 9 concentration of dye is 5x10-6 M). To stop the labelling reaction 1 mL of free EVs-FBS (previously ultracentrifuged at 100,000 g during 16h at 4ºC to remove exosomes) was added for 1 minute. To wash the excess of dye from EVs, the tube of EVs suspension was filled with M199 media with 5% FBS (EVs-free) and twice ultracentrifuged at 100,000 g, 4°C, for 30 min. The final pellet was resuspended in 100µL of embryo culture medium (SOF) as described below with 5% FBS (EVs-free) and used to prepare the drops for embryo development.

In vitro produced embryos at the blastocyst stage (with intact zona pellucida) and hatched embryos (with total or partial absence of zona pellucida) were in vitro cultured with green-labeled EVs or control (dye-PBS) For this experiment, 4 replicates were performed, with 15-20 embryos incubated with green-EVs or control (dye-PBS) for each replicate.

Proteomic analysis

Mass Spectrometry (MS) analysis

EVs preparations from in vivo and in vitro origins were analyzed by SDS-PAGE combined with nanoLC-MS/MS with spectral counting and extracted ion chromatography (XIC) methods of quantification.

Sample preparation for MS analysis

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk.

Data mining and Bioinformatics analysis

Gene symbols and Entrez Gene IDs (bovine and putative human orthologs) were mapped for all protein identifications and analyzed using the online bioinformatics tools Having in mind the practical application of EVs in the IVP lab, we decided to compare the effect of fresh and frozen in vivo EVs supplementation versus control (nonsupplementation) in our embryo culture system. Experiments were performed in 4 replicates. EVs were prepared pooling oviducts from 3 animals for each replicate as above mentioned. For each replicate, EVs preparations were divided in two aliquots

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 16 16

(frozen and fresh samples). Frozen samples were kept at -80 ºC 3h while fresh samples were kept at 4 ºC until IVC media supplementation. For each replicate, EVs protein concentration was measured ranging from 1.61-5.34 mg/mL and EVs supplementation was added to the culture medium at a final concentration of 0.22-0.42 mg/mL. Fresh and frozen EVs were diluted in IVC media and filtered (0.22µm). Then 25 µL drops of IVC medium supplemented with or without EVs were prepared. Subsequently, groups of 25 presumptive zygotes were cultured into these 25 µL drops of SOF medium with or without EVs supplementation, overlaid with 700 mL of mineral oil. Embryo-EVs coculture was performed into 38.8 C, 5% O2, 5% CO2, and 90% N2 conditions during 9 days. Embryos were allocated in 3 groups according to the experimental design:

control, fresh EVs and frozen EVs to evaluate embryo development and quality in terms of cleavage (Day 2 post IVF), blastocyst rate (Days 6, 7, 8 and 9), hatching rates (Days 8 and 9) and number of cells (Day 9).

RESULTS

Differential protein profile in oviduct EVs from in vivo and in vitro origin

We validated the presence of EVs in both in vivo and in vitro samples by using TEM as well as Western blot analysis. Transmission Electron microscopy (TEM) observations confirmed the presence of EVs in bovine oviduct flushings (Figure 1A) and also in the conditioned media from BOEC primary in vitro culture (Figure 1B). All four replicates analyzed from in vivo and in vitro preparations showed a population of smallextracellular vesicles (30-100nm) resembling exosomes and a population of largeextracellular vesicles (>100nm-) resembling microvesicles (Figure 2A and 2 B). In the literature, microvesicles ranged from (>100 up to ∼1,000 nm) (Raposo & Stoorvogel 2 showed the distribution of exosomes and microvesicles in the in vivo (Figure 2A) and in vitro preparations (Figure 2B).

Western blotting for exosomal protein markers and oviduct proteins with known reproductive roles were performed in EVs in vivo and in vitro preparations and their cells of origin (Figure 3). EVs were positive for HSP70, a recognized exosomal protein (positive control) present in 89% of exosome proteomic studies (Mathivanan et al. 2010, Klohonatz et al. 2016) and negative for Grp78, an endoplasmic reticulum marker detected in BOEC from both origins and not in EVs (negative control) (Figure 3).

Moreover, in vivo EVs expressed oviduct glycoprotein (OVGP1), heat shock protein A8 (HSPA8) and myosin 9 (MYH9), while only HSPA8 and MYH9 were detected in in vitro EVs. When the cells of origin were analyzed for these reproductive proteins, OVGP1, MYH9 and HSPA8 were expressed in all in vivo samples. In contrast, MYH9 and HSPA8 were expressed in all in vitro oviduct cells whereas OVGP1 was not expressed in any of the samples in vitro. Taken together, the Western blot (molecular) data and TEM (bio-physical) showed that both in vivo and in vitro oviduct cells produce EVs but with different qualitative and quantitative characteristics.

To understand the potential roles of EVs in the oviduct environment as modulators of gamete/embryo-oviduct cross-talk, we performed the first proteomic analysis of oviduct EVs using Mass spectrometry. This analysis allowed us to compare the in vivo and in vitro oviduct EVs signature. Mass Spectrometry identified a total of 315 proteins, from which 97 were exclusively detected in in vivo EVs, 47 were found only in in vitro and 175 were in common to both samples (Venn diagram, Figure 4, and Supplementary data S1-S3). Moreover, Figure 4 represents the total of proteins identified in in vivo (270) (GO) analysis using PANTHER database revealed that a high number of these proteins was involved in metabolism (24%), cellular process (20.4%), localization (10.2%), developmental processes (7.7%), immune system (5.6%), response to stimulus (7.4%), and in reproductive processes (1.4%) (Figure 6). Further analysis revealed that more than 58% of EVs proteins involved in cellular process were associated with cell-to-cell communication (Figure 6). Moreover, DAVID functional annotation clustering for EVs proteins showed that clusters related to "vesicle", "cytoplasmic vesicle", and "membrane-bounded vesicle" had the highest enrichment score (enrichment score 19.9, 62 proteins involved) while clusters related to fertilization were also found with a relative high enrichment score (

ClueGO) was used. Figure 7 shows clear differential networks of functional categories for in vivo and in vitro EVs proteins.

3. Demonstrating the traffic of oviduct EVs to the early embryo.

Here, we demonstrated that IVP embryos were able to internalize in vivo EVs. We selected in vivo EVs for our experiment, since our proteomic analysis pointed out important differences between in vivo and in vitro exosomes such as OVGP1 protein, only present in in vivo EVs and involved in supporting early embryo development.

EVs were isolated and labeled with green fluorescent dye (PKH67), filtered (0.

Oviduct EVs supplementation improved bovine in vitro blastocyst yield and quality

Having in mind the practical application of EVs in the IVP lab, we decided to compare the effect of fresh and frozen in vivo EVs supplementation versus control (nonsupplementation) in our embryo culture system (4 replicates). EVs supplementation had no effect on embryo cleavage (day 2) (Figure 9A) (73.58±3.44; 76.57±1.92 and 81.04±3.64 for fresh, frozen, and control, respectively) but influenced blastocyst rates over time (day 7-9) (Figure 9B). Interestingly, frozen EVs significantly improved 9B). Fresh EVs showed a significant effect on embryo development at day 9 compared to the control (day 9: 23.78±4.01;

35.2±4.86 and 49.36±0.64, Figure 9). Embryo quality was measured in terms of the hatching ability and number of cell/blastocyst. There were no differences on hatching rates among all groups at day 8 while the addition of frozen EVs improved significantly the hatching rate at day 9 (Figure 9C) from 7.5% to 26%. The total number of cells was also improved by frozen EVs addition (Figure 9D). Our results showed that oviduct EVs supplementation during in vitro embryo development improves blastocyst yield, quality and extends embryo survival overtime.

DISCUSSION

Our study demonstrated that EVs are essential components of oviduct secretions from in vivo and in vitro origins. Moreover, our results provide with the first oviduct EVs signature. We found differential protein profiles between in vivo and in vitro EVs, under our experimental conditions. We demonstrated that EVs from in vivo origin were up taken by in vitro produced embryos and exert a functional effect by enhancing flushings´ obtained were immediately centrifuged at 300xg, 15 min followed by 12000xg 15 min. Then, BOEC cells were isolated, seeded and in vitro cultured for 14 days, as in our previous studies. It is possible that EVs collected from oviducts immediately post-mortem or transported under conditions different to those in our study (i.e ice) could differ in EVs quality or protein abundance, despite EVs content seems well-preserved. On the other hand, the use of a different culture system, with distinct media, period of cell culture could also affect EVs production/content. To the best of our knowledge, there is not any available study that has shown to which extend in vitro culture can affect the EVs production and content compared to in vivo EVs. Even more, no information exists regarding if the days of in vitro culture, the media or other in vitro factors can change their content. Cell culture models are important tools for revealing specific effects and mechanisms of cell populations and currently are being used for many studies as a source to obtain EVs. However, it is extensively known that it is very difficult to find an in vitro cell culture model that allows the cells to be as much as possible similar to the in vivo ones (in regard to gene expression, secretion, EVs etc.).

Recently, two novel culture methods for oviductal cells based on air-liquid interphase culture system (Chen et al. 2017) and a 3D-printing oviduct device (Ferraz et al. 2017) have been proposed to generate oviduct fluid surrogates more similar to the in vivo ones. Therefore, it is possible that using these novel systems, the in vitro content of EVs could be more similar to the in vivo ones.

In the present study, the proteomic profiling of oviduct EVs showed an exosome signature and confirmed that oviduct exosomes contain basic machinery important for biogenesis, trafficking, fusion and release. However, our TEM observations also revealed the presence of microvesicles. Establish methods that allow to discriminate et al. 2000). Our results support the hypothesis that exosomes/microvesicles could be one of the mechanism(s) by which BOEC may release these proteins into the oviduct milieu and transport them to the embryo, allowing successful gamete interactions and subsequently early embryonic development.

Among the different reproductive proteins identified in oviduct EV under our experimental conditions, we would like to highlight the presence of OVGP1, MYH9, HSP90 (in its HSP90B1, HSP90AA1, HSP90AB1 forms) and lactadherin (PAS6/7 or MFGE8) because of their important functions in gamete/embryo-oviduct crosstalk.

OVGP1 is the major non-serum protein present in the oviduct fluid in different species Sutton et al. 1984, Buhi et al. 1990). It increases sperm viability and motility [START_REF] Abe | Bovine oviduct-specific glycoprotein: a potent factor for maintenance of viability and motility of bovine spermatozoa in vitro[END_REF]; modulates sperm capacitation and fertilization (King et al. 1994) and enhances development rates (Kouba et al. 2000). Furthermore, OVGP1 seems to bind to both gametes through the interaction of its non-glycosylated N-terminal conserved region with MYH9 (Kadam et al. 2006). Our results imply that both proteins might be secreted via EVs, or at least partly. Since both OVGP1 and MYH9 were detected in vivo EVs, while only MYH9 was expressed in vitro EVs. The fact that OVGP1 is only expressed in in vivo exosomes/microvesicles was not surprising since OVGP1 is expressed in BOEC after collection but its mRNA is strongly decreased after 7-10 days of culture. Considering that OVGP1 is secreted under steroid control, it is possible that the lack of hormonal stimulation in our experiment could explain the absence of OVGP1 in the in vitro exosomes/microvesicles. Together with OVGP1, HSP90B1 was also expressed in oviduct EVs and is associated to ZP hardening mechanism (Mondejar et al. 2012). HSP90 has been shown on the surface of 25% of the live capacitated sperm population that is capable of interacting with the ZP of the oocyte [START_REF] Asquith | Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition[END_REF]).

In addition, we identified lactadherin in EVs preparations, a protein common in Having in mind the practical application of EVs in optimizing IVF systems, we compared the effect of fresh and frozen EVs during in vitro embryo development.

Despite both fresh and frozen EVs had a positive effect on embryo development, surprisingly the use of frozen EVs showed better results for IVP. To the best of our knowledge, no studies have been performed to evaluate properly the impact of the freezing procedure on the EVs structure and/or content that could explain our results.

The information available to date in the literature is controversial with different studies

showing conflicting results regarding the resistance of EVs to freezing (Bosch et al. process on EVs, our data provide clear evidence of the positive effect of fresh and frozen oviduct EVs on embryo development. Therefore, our study points out that the use of oviduct EVs is a good strategy to optimize in vitro embryo production. Further studies will be conducted to evaluate whether EVs can also improve the pregnancy outcomes after transferring embryos co-incubated with EVs, making them potential tools for the application of other biotechnologies.

In summary, our study identified the first oviduct-derived EVs protein signature, and reveals a set of proteins with important roles in gamete/embryo oviduct interactions that have not been previously identified in the oviduct EVs cargo. Moreover, our results highlight the differential protein cargo between in vivo and in vitro EVs. Information of great importance since most of functional studies of EVs are based on EVs derived from cell lines or primary culture and extrapolated to in vivo EVs biology and function.

Functionally, we demonstrated that EVs were internalized by the embryo during in vitro culture and enhance their ability to reach the blastocyst stage, to hatch and increase their survival overtime. Taken together, this study brings new insights into the contribution of EVs as modulators of the oviduct-gamete/embryo crosstalk and opens up a new avenue for the use of EVs to optimize ART in human and livestock species.

CONFLICT OF INTEREST

None declared.

FUNDING

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 ( 4 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054

Figure 3. Western blotting characterization of bovine oviduct EVs from in vivo and in vitro origin for known exosomal markers and oviduct proteins with known reproductive roles. i) Both EVs preparations were positive for HSP70, a known exosomal protein marker and negative for Grp78, an endoplasmic reticulum marker detected on BOEC; ii) in vivo EVs expressed oviduct glycoprotein (OVGP), heat shock protein A8 (HSPA8) and myosin 9 (MYH9), while only HSPA8 and MYH9 were detected in in vitro EVs; iii) When the cells of origin were analyzed for these reproductive proteins, OVGP1, MYH9 and HSPA8 were expressed in all in vivo samples, but only MYH9 and HSPA8 were expressed in in vitro oviduct cells.

162x292mm (300 x 300 DPI)

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. ( 2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054

(

  RT) were used to perform extracted ion chromatograms (XICs). The framing parameters were set at 0.02 Da for the 300-1800 m/z mass range and 6 min for the RT window for all MS2 data. The autodigested tryptic peptide at m/z 1082.0300 was used to normalize independent samples. The algorithm determined peptide abundance between 2 sample groups, frame-by-frame. A t-test was performed to characterize changes between in vivo and in vitro EVs preparations. Differences were considered statistically significant at p-value<0.01. Following the Proteome Discoverer version 1.3. databank searches (Thermo Fisher Scientific) using the Mascot server, the .msf files were integrated into SIEVE. The results were filtered with protein normalized ratios < 0.5 and ratio > 2, with Mascot ion scores >20. Identified frames were accepted manually when peptides were validated by the Protein and Peptide Prophet Algorithms used in Scaffold software. MS data have been deposited to the ProteomeXchange Consortium (Vizcaino et al. 2014) via the PRIDE partner repository with the dataset identifier 10.6019/PXD002280.

  available via the biological DataBase network bioDBnet (tool db2db) (http://biodbnet.abcc.ncifcrf.gov/;(Mudunuri et al. 2009)) and custom tools integrated in a local Galaxy(Giardine et al. 2005) installation (NCBI annotation mapper, Mammalian Ortholog and Annotation database, MOADb; Bick J, ETH Zurich, Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 14 14 unpublished results 2016). The background dataset for the analysis was the human genome. To obtain meaningful findings of the EV proteins identified functional analysis was performed using PANTHER (http://www.pantherdb.org; (Thomas et al. 2003)) and DAVID Functional Annotation (https://david.ncifcrf.gov); (Huang et al. 2007)). To visualize interactions among candidate EV proteins and integration of the different networks the Cytoscape app ClueGO was used (http://www.cytoscape.org/; Cytoscape 3.3.0 and app ClueGO v2.2.5; (Shannon et al. 2003)). Western blotting Proteins were separated by SDS-PAGE (8-16% gradient polyacrylamide gels using 4 µg of proteins per lane) and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences Whatman TM ) over 16h at 30v, 300A. The membranes were washed in distilled water and blocked with Tris buffered saline (TBS) containing Tween 20 (0.5% (w/v)), and supplemented with lyophilized low-fat milk (5% w/v) for 1h at room temperature. The membranes were incubated with primary antibodies diluted in TBS-Tween containing low-fat milk (1% w/v) for 2h at 37ºC with gently shaking. The primary antibodies used were: anti-heat shock protein 70 (HSP70; Stressgen, SPA-810); anti-78 kDa glucose-regulated protein (GRP78; Santa Cruz Biotechnology sc-13968); anti-heat shock protein A8 (HSPA8; bioss.com, bs-5117R); anti-bovine oviduct glycoprotein (OVGP; kindly donated by P.A. Mavrogianis at University of Illinois in USA); anti-Myosin heavy chain 9 (MYH9 (H40), Santa Cruz Biotechnology, sc-98978); anti-Cluster of Differentiation 109 (CD109, Santa Cruz Biotechnology, sc98793); and anti-Lactadherin (PAS6/7, a gift from Dr. J.T. Rasmussen). After primary antibodies incubation, the membranes were washed with TBS with 0.5% Tween 20 and incubated overnight at 4ºC under agitation with secondary antibodies. The secondary antibodies Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 15 15 used were: horseradish peroxidase (HRP)-anti-mouse (Sigma A4416) or anti-rabbit (Sigma A6154). Blots were developed using a mixture of two chemiluminescence substrates developing kit (GE Healthcare AmershamTH ECL SelectTH Western blotting detection Reagent RPN2235 and Supersignal West Pico #34087 Chemiluminescent Substrate Thermo Scientific). In vitro embryo production (IVP) and EVs supplementation to in vitro embryo culture Bovine embryos were produced in vitro as previously described by Cordova and colleagues (Cordova et al. 2014). Briefly, bovine ovaries were collected at a local slaughterhouse. Cumulus-oocyte complexes (COC) were aspirate, washed and incubated in maturation media for 22 hours. Subsequently, COC were fertilized with semen from a bull with proven fertility. Twenty hours after IVF, presumptive zygotes were vortexed to remove cumulus cells and attached spermatozoa and washed into wells containing 500 µL of in vitro culture medium. The medium used for in vitro culture (IVC) was synthetic oviduct fluid (SOF) medium (Holm et al., 1999) supplemented with 5% fetal calf serum (FCS, MP Biomedicals, MP5418) (EVs-depleted by ultracentrifugation).

  22μm) and co-incubated with blastocysts (with intact zona pellucida) and hatching/hatched (H) blastocysts (with partial or total absence of pellucida) produced in vitro. Confocal microscopy observations confirmed that in vivo EVs were internalized by blastocysts (Figure 8, A-C) and H-blastocysts (Figure, D-F) and located around the nucleus. Figure 8 (I-J) shows that EVs were actually present in the cytoplasm and not only attached to the embryo membrane. No green fluorescent EVs were observed in the negative controls of embryo co-cultured with PBS-dye (G-H).

  embryo development and quality during in vitro culture.TEM observations and WB from our study confirmed the presence of EVs in oviduct secretions from in vivo and in vitro origin. Distribution size by TEM measurements showed different populations of exosomes and microvesicles from both sources of samples ranging from 30-250nm. Previous analysis performed by laboratory using dynamic light scattering analysis (DLS) showed a higher abundance of bigger vesicles than exosomes in EVs conditioned media when compared to oviduct fluid(Alminana C Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 22 22 2014) in contrast to current measurement by TEM. We believe that DLS results could be due to artefacts, measurements of aggregates of vesicles instead of individual vesicles (Muller et al. 2014). Moreover, Van del Pol et al., showed that different techniques can give a different size distribution and a different concentration for the same vesicle sample (van der Pol et al. 2014). Nevertheless, our results are in agreement with previous studies isolating exosomes and microvesicles from bovine oviduct fluid following a protocol similar to us (centrifugation at 100,000 xg) (Lopera-Vasquez et al. 2017). Exosomes and microvesicles were also identified in both oviduct fluid and BOEC derived conditioned media by Al-Dossary et al., (Al-Dossary et al. 2013) and Lopera-Vásquez et al., (Lopera-Vasquez et al. 2016), respectively. Our study differs from those in 2 important aspects: 1) our study used a wide proteomic approach to characterize oviduct EVs content and decipher the role of EVs during early gamete/embryo-maternal interactions; while Al-Dossary and colleagues (Al-Dossary et al. 2013) focused their study on a specific protein approach contained in murine Oviduct EVs, PMCA4a, which is essential for sperm hyperactivated motility and fertility; and 2) we compared in vitro and in vivo EVs obtained from conditioned media of primary culture of BOEC and oviduct fluid from same oviducts at the early post ovulatory stage (1-4 after ovulation) to minimize variability. Lopera-Vásquez et al., used oviducts at the mid-luteal phase of the estrous cycle (Lopera-Vasquez et al. 2016). Greening et al., demonstrated that different hormonal environment during the estrous cycle modulate content of human endometrial derived EVs (Greening et al., 2016). Our findings suggest that BOEC in vivo might not secrete the same population of exosomes/microvesicles than in vitro under our experimental conditions. It is possible Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 23 23 that the in vitro culture of BOEC may affect their ability to secrete and release exosome/microvesicles and their content. BOEC in monolayer after several days in culture are less likely to mimic the oviduct environment than BOEC in vivo, as reflected by the loss of morphological hallmarks such as cilia and secretory granules (Rottmayeret al. 2006). Differences between in vivo and in vitro exosome populations were also reported byOstman et al., (Ostman et al. 2005), when comparing exosomes secreted by in vitro propagated tumor cells versus tumor cells grown in vivo. Here, we showed quantitative and qualitative proteomic differences between EVs content from in vivo and in vitro origin by MS analysis. Moreover, our functional analysis of the EVs proteins from both sources showed that they were associated to different biological functions and networks (Figure7). Altogether, highlights the distinct functionality of in vivo and in vitro EVs during oviduct-embryo interactions. Since EVs play a role in cellto cell communication through the transfer of their cargo, it can be expected that the different proteomic oviduct EVs composition between in vivo and in vitro may exert distinct functional effects on embryo(s) and gametes.It is worthy to mention that the experimental conditions used in the present study (oviduct source, oviduct collection, BOEC collection, in vitro cell culture system, period of cell incubation and culture media, etc) might affect the in vivo and/or in vitro EVs secretion/content identified here. To obtain the oviductal flushing's the conditioned media from BOEC culture, we used a protocol developed in our laboratory that has shown to be a good method to prepare BOEC monolayer to support embryo development but also, to study the embryo-oviduct dialogue, and analysing the BOEC gene expression (Schmaltz-Panneau et al. 2014). To avoid that the in vivo "debris" and "apoptotic/dead cells" contained in the oviduct fluid could affect the in vivo EVs, Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 24 24

  exosomes studies and involved in ZP binding. It has been previously identified in vitro microvesicles released by endometrial cell cultures under hormonal stimulation(Sarhan et al. 2013). However, lactadherin secretion in vitro has been also associated to unhealthy cells(Delcayre & Le Pecq 2006), despite in our in vitro culture system 90% of BOEC were viable. It is interesting to note that exosomes are packing many proteins from heat shock protein 70 and 90 families as well as other proteins involved in free radicals scavenging(peroxiredoxins, thioredoxin, etc). These contents may bring to the gametes and embryos some additional factors to survive in the in vivo and in vitro environment.Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction(Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 27 27 Considering all the embryotrophic factors contained in the EVs, we evaluated whether EVs could be up taken by the embryo and exert a functional effect on embryos. Our results showed that oviduct EVs were internalized by the embryo being capable of passing through the zona pellucida and being located around the nucleus of most embryonic cells. Our results are in line with other studies showing the uptake of uterine EVs by the embryo/conceptus at later stages (Vilella et al. 2015, Burns et al. 2016) (Greening et al. 2016, Bidarimath et al. 2017). Burns et al. suggested that the uterine EVs uptake could have an essential role in elongation of the conceptus (Burns et al. 2016). Vilella and colleagues (Vilella et al. 2015) demonstrated that Hsa-miR-30d, contained in uterine exosomes, could induce transcriptional and functional modifications in the adhesive competence of the embryo. In our study, we provide strong evidence for the functional effect of oviduct EVs in supporting bovine preimplantation embryo development, since EVs supplementation improved embryo development and embryo quality. Our results are in part in line with Lopera-Vásquez etal., (Lopera-Vasquez et al. 2016, Lopera-Vasquez et al. 2017), reporting that the use of in vitro frozen/thawed BOEC-derived EVs improve embryo quality, by increasing the number of cells and the survival of blastocyst after cryopreservation. However, these authors did not report any improvement in embryo development over time or hatching rates during in vitro culture, as our study shows. The distinct effect of EVs on embryo during IVC between studies could be explained by the differences found in the protein content between EVs from in vivo and in vitro origin or other EVs molecular components (mRNA, miRNA). Moreover, the moment of the estrous cycle or the different parts of the oviduct (Ampulla, Ithsmus) from which they were collected could also have an effect(Greening et al. 2016, Lopera-Vasquez et al. 2017). Our next studiesComment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 28 28 will be focused in further analyzing the content of EVs at mRNA and miRNA across the estrous cycle and investigating the possible epigenetic effects of EVs on preimplantation embryo development.

  2016). Most of studies are based on fresh or frozen samples (blood, fluid, urine) from which EVs are isolated. A few studies have indicated that storing samples at -80ºC do not alter EVs morphology or size (Sokolova et al. 2011, Sarker et al. 2014). While others have suggested that freezing may induce membrane damage and leakage of EVs content in the absence of perceivable changes of size and concentration. A recent study has found a significant reduction in the bi-layer membrane of frozen vesicles (-80ºC) when compared to fresh EVs (Teng et al. 2015), which could explain our results. Regarding their content and functionality, studies point out that freezing seem to preserve almost completely EVs associated proteins (Zhou et al. Kidney Int. 2006 Apr; 69(8): 1471-1476) and do not impair their functionality (Sokolova et al. 2011, Jayachandran et al. 2012) as we have observed in our study. The alterations of EVs following freezing remains a matter of debate in the field of extracellular vesicle research. Although further studies are required to elucidate the impact on the freezing Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 29 29

1 FIGURE LEGENDS Figure 1 .

 1LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Histograms showing the size distribution of bovine oviduct EVs in in vivo

Figure 3 . 2 Figure 4 .

 324 Figure 3. Western blotting characterization of bovine oviduct EVs from in vivo and

Figure 5 .

 5 Figure 5. Validation of Mass Spectrometry analysis of bovine oviduct EVs by

Figure 6 .

 6 Figure 6. Functional analysis of bovine oviduct EVs proteins identified by Mass

Figure 7 .

 7 Figure 7. Integrative visualization of the differential proteins identified in in vivo

Figure 8 .3

 8 Figure 8. Uptake of in vivo EVs by in vitro produced embryos. EVs preparations

Figure 9 .

 9 Figure 9. Effect of fresh and frozen in vivo EVs on the embryo development and

Figure 1 .

 1 Figure 1. Transmission electron microscopy observations of bovine oviduct EVs preparations from in vivo (A) and in vitro (B) origin. Structures pointed by blue arrows with 30-100nm size resembling to exosomes and structures pointed by red arrows > 100nm size resembling to microvesicles were identified in oviduct flushing (in vivo; A) and conditioned media from BOEC culture (in vitro; B) preparations. 69x29mm (300 x 300 DPI)

Figure 2 .

 2 Figure 2. Histograms showing the size distribution of bovine oviduct EVs in in vivo and in vitro preparations.Four replicates of EVs from in vivo (A) and in vitro (B) preparations were analyzed using TEM and measurement of vesicles were performed. Populations of exosomes (30-100nm) and microvesicles (>100nm-250) (Figure2Aand 2 B) were found in both EVs preparations.

Figure 4 .

 4 Figure 4. Venn diagram showing the number of bovine oviduct EVs proteins identified exclusively in in vivo and in vitro preparations and in common. Venn illustrates that 97 proteins were exclusively detected in in vivo EVs, while 47 were found only in in vitro and 175 were in common to both samples. 119x93mm (300 x 300 DPI)

Figure 5 .

 5 Figure 5. Validation of Mass Spectrometry analysis of bovine oviduct EVs by Western immunoblotting. Western blot analysis confirmed MS results in the 4 biological replicates for CD109 and Lactadherin. CD109 wa only expressed in in vivo EVs while Lactadherin was expressed only in in vitro EVs.

Figure 6 .

 6 Figure 6. Functional analysis of bovine oviduct EVs proteins identified by Mass Spectrometry by PANTHERdatabase. EVs proteins isolated from in vivo and in vitro preparations were subjected to ontology and pathway analysis using PANTHER and Gene ontology algorithms and subsequently classified based on their Biological Process.

Figure 7 .

 7 Figure 7. Integrative visualization of the differential proteins identified in in vivo (red) and in vitro (green) bovine oviduct EVs and their biological functions and protein interactions using Cytoscape app ClueGO. Differential networks of functional categories from in vivo (red) and in vitro (green) EVs proteins are shown.

Figure 8 .

 8 Figure 8. Uptake of in vivo EVs by in vitro produced embryos. EVs preparations from in vivo origin were labelled with green fluorescent dye (PKH67), filtered (0.22µm) and co-incubated with blastocysts (A-C) and hatching/hatched (D-F) blastocysts. Embryos co-cultured with green-labelled EVs were fixed and stained with Hoechst 3342 to visualize the nucleous and with Actin Red phallodin to visualize the membrane of the cells. Fluorescence (A-F) images demonstrating active uptake of green-labelled Evs by embryos in vitro during culture. No green fluorescent exosomes/microvesicles were observed in the negative controls of embryo co-cultured with PBS (G-H, Controls). Detail of EVs inside the blastocyst cells, confirmed that in vivo EVs were not only attached to the embryo membrane but were actually internalized by these cells and were present in the cytoplasm (I-J). Images were obtained with 20X with an additional zoom factor from 1 to 2.8X. Scale bar = 50µm.

Figure 9 .

 9 Figure 9. Effect of fresh and frozen in vivo EVs on the embryo development and the quality of in vitro produced embryos. Embryo were co-incubated with EVs for 9 days after IVF (4 replicates, number of initial oocytes for Control n=353; Fresh EVs n=332 and Frozen EVs n=332). Cleavage (A), Blastocyst (B) and Hatched blastocyst (C) rates were expressed as percentages (Mean ±SEM) and were calculated on initial oocytes numbers. Number of cells /blastocyst is shown in figure section (D). Different letters in the graphs represent significant differences (P<0,05). Comparison among treatments in blastocyst rates are made on the same day of culture.

oviduct epithelial cell in vitro culture

  Our BOEC in vitro culture system has already been used to study early oviduct-embryo interactions, demonstrating to be a good oviduct-like environment to support embryo

	development in vitro (Cordova et al. 2014, Schmaltz-Panneau et al. 2014). BOEC were
	cultured in 25 cm2 flasks (FALCON 25cm2 353109) with TCM 199 (Sigma M4530,
	France) supplemented with 10% Fetal Bovine Serum (Sigma F9665, France) and
	gentamycin (Sigma G1272, 10 mg/mL) in a humidified atmosphere with 5% CO2 at
	38.8 ºC. The medium was completely renewed at day 2. Subsequently, half of the

Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 7 7 Bovine medium was replaced every two days until cells reached confluence (6-8 days). Then, BOEC were washed and cultured in TCM-199 free of serum. After two days, the serum free medium was completely renewed and the cells were cultured for two additional days before collection of conditioned medium. BOEC viability was determined after collection of conditioned media by using Live/Dead viability assay kit (LIVE/DEAD® Cell Viability Assay, Life Technologies, L3224). At this point, an aliquot of in vitro BOEC was stored at -20ºC for further comparison of protein content with in vivo BOEC (day 0) and EVs collected from them by Western blotting.

  EVs samples. While the bar graph below shows the number of proteins in common (175) or exclusive to in vivo or in vitro EVs (142). From the total of 315 identified proteins, 186 were differentially expressed when in vivo and in vitro

	19
	HSPA5, HSPA8, HSP90AA1, HSP90AB1, HSP90B1) and other proteins related to
	adhesion such as MFGE8/Lactadherin and integrins (ITGB1).
	EVs were compared (p-value<0.01; ratio<0.5 or ratio>2) (Supplementary data S4). Besides the identification of common exosomal proteins, we performed functional
	analysis of identified EV proteins from in vivo and in vitro origin to obtain a better
	understanding of the role of the EV proteins in different biological processes and,
	Mass Spectrometry results were confirmed by Western blot analysis on candidate
	particularly, in gamete/embryo-oviduct communication and in supporting embryo
	proteins present in EVs and associated to reproductive functions (Figure 5). Western
	development. From the 315 proteins identified in EVs preparations, Gene ontology
	blot analysis was performed in 4 independent biological replicates for CD109, and
	Rab GTPases (RAB5C, RAB7A, RAB11B, RAB1A; ARF4). The identified tetraspanin
	and rab family proteins were present in both in vivo and in vitro EVs. In addition, we
	found that EVs cargo were enriched in Annexins, another class of proteins commonly
	seen in exosomes, involved in membrane trafficking and fusion events (ANXA1,
	ANXA2, ANXA3, ANXA4, ANXA5, ANXA7, ANXA8, ANXA11). Most of them
	19

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 18 18 and in vitro (222) Lactadherin. While Lactadherin was expressed only in in vitro EVs preparations, CD109 was expressed only in in vivo EVs preparations. These results confirmed the different proteomic profile of EVs from in vivo and in vitro origin.

2. Deciphering the EV proteomic content from in vivo and in vitro origin

Mass Spectrometry data provided the first proteomic signature of oviduct EVs, and identified family of proteins in their cargo characteristic of exosome protein composition. The exosomal cargo included proteins involved in exosome biogenesis and intracellular vesicle trafficking, including tetraspanins (CD46, CD109, CD9) and common to both EVs preparations except ANXA7 that was only expressed in in vivo

EVs. EVs were also enriched with heat-shock proteins (HSPA1A, HSP60, HSPA4,

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (

2017

). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054

  Table I). A complete list of clusters is provided in

	Supplementary data S5.
	A deeper analysis combining PANTHER, DAVID and GeneCards databases and the
	literature, revealed 36 EVs proteins (11.42% from 315) involved in important
	reproductive functions such as fertilization and embryo development (Table II).
	Furthermore, to obtain a more integrative visualization of the differential proteins

identified in in vivo and in vitro EVs and their biological functions Cytoscape (app Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (

2017

). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 20

  at days 7 and 8 compared to fresh EVs and control (day 7: 32.99±3.13; 30.18±3.99 and 40.91±2.61 and day 8: 34.04±2.67; 37.92±2.57 and 45.91±1.12 for control, fresh and frozen, respectively) (Figure

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (2017). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054 21 21 blastocyst rates

Table I .

 I Selected results of DAVID functional annotation clustering for proteins 2 identified in bovine oviduct EVs from in vivo and in vitro origin. 3 Actin cytoskeleton organization (21, 4.5); Actin filament-based process (23,4.6)ATP binding (61, 1.9); Adenyl ribonucleotide binding (61, 1.9); Purine nucleoside binding (61, 1.8);Glucose catabolic process (11, 9.1); Hexose catabolic process (11, 7.7); Glycolysis (9, 9.2) Vesicle lumen (9, 9.1); Cytoplasmic membrane-bounded vesicle lumen;(8, 8.5) Regulation of apoptosis (36, 2.1); regulation of programmed cell death (36, 2.1); Regulation of cell death (36, 2.1) Anti-apoptosis (16, 3.7); negative regulation of apoptosis (21, 2.9); negative regulation of programmed cell death (21, 2.8); negative regulation of cell death(21, 2.8)

				2
	SEC23IP	SEC23-interacting protein [Bos taurus]	Spermatid development	GeneCards
	PDXK	pyridoxal kinase [Bos taurus]	Epididymis Secretory Sperm Binding Protein	GeneCards
	DCXR	L-xylulose reductase [Bos taurus]	Sperm surface protein	GeneCards
	ATP5A1	Chain A, The Structure Of F1-Atpase Inhibited By	Epididymis Secretory Sperm Binding Protein,	GeneCards
		Resveratrol.	EMBRYO DEVELOPMENT
	Representative functional terms of overrepresented annotation clusters HSPA4 heat shock protein family A (Hsp70) member 4	Sperm fertilizing ability	Enrichm. Literature	No.
	HSPE1	PREDICTED: 10 kDa heat shock protein,	Early Pregnancy factor	score 1 Literature	Proteins
	Annotation cluster for proteins identified in exosomes from in vivo and in vitro origin mitochondrial-like [Macaca mulatta]
	Vesicle (62, 4.3); cytoplasmic vesicle (57, 4.1); membrane-bounded vesicle (54, 4.4) HSPA8 heat shock protein family A (Hsp70) member 8	sperm fertilizing ability	19.9 Literature
	Ribosome (21, 17.4); translational elongation (25, 11.9); protein biosynthesis (26, 8.3) HSP90B1 heat shock protein 90kDa beta family member 1	Sperm fertilizing ability	14.3 Literature
	Nucleotide-binding (78, 2.8); Purine ribonucleotide binding (80, 2.1) HSP90AA1 heat shock protein 90kDa alpha family class A	Sperm fertilizing ability	10.9 Literature
	Annexin (8, 34.6); Annexin 3; Annexin 4; Annexin 1; Annexin 2 member 1		8.5
	HSP90AB1	heat shock protein 90kDa alpha family class B	Sperm fertilizing ability	6.8 Literature
		member 1		6.6
	ANXA2	annexin A2	Embryo adhesiveness to endometrium, sperm-	5.0 Literature
			oviduct binding	5.0
	ANXA5	annexin A5	Formation Sperm reservoir, sperm-oviduct	4.6 Literature
			interaction	4.3	21
	4
	Phospholipase inhibitor activity (5, 19.9); Lipase inhibitor activity (5, 15.9)		4.2
	Myosin (8, 9.5); Myosin complex (8, 5. 7)		3.9
	Heat shock protein Hsp70 (5, 22.45); Chaperone HSP70 (4, 17.2)		3.8
	Primary lysosome (3, 35); Specific granule (3, 20)		2.4
	Hemostasis (10, 4.5); Regulation of body fluid levels (9, 3.7); Blood coagulation (9, 4.2); Wound healing (11, 2.8)	2.9	13
	Cell migration (15, 2.6); Localization of cell (15, 2.5); Cell motility (15, 2.5)		2.7	16
	Heat shock protein Hsp90 (3, 32.1)		1.7
	Peroxiredoxin activity (4, 23.9); Response to reactive oxygen species (5, 3.2); Antioxidant activity (4, 4.07)	1.8
	Calcium-binding region:1; low affinity (4, 13.4); Calcium-binding region:2; high affinity (4, 11.5)	2.1
	Fertilization (5, 20.2); Single fertilization (5, 4.0)		2.2
	Cellular ion homeostasis (16, 2.1); Chemical homeostasis (17, 1.6);		1.7	17
	Actin capping (4, 12.8); Negative regulation of cytoskeleton organization (5, 4.4); Negative regulation of organelle organization (5, 2.9)	1.7
	1 geometric mean of member's p-values of the corresponding annotation cluster (in -log10 scale); 2 in brackets:
				2

4

number of genes and fold enrichment of the functional term.

5

Comment citer ce document : Alminana Brines, C., Corbin, E., Tsikis, G., Soares De Alcantara Neto, A., Labas, V., Reynaud, K., Galio, L., Uzbekov, R., Garanina, A. S., Druart, X., Mermillod, P. (

2017

). Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. Reproduction (Cambridge), 154 (4), 253-268. , DOI : 10.1530/REP-17-0054

ACKNOWLEDGEMENTS

We thank Christine Longin (GABI, INRA-UMR 1313) for technical assistance with Electron Microscopy.

We thank Dr. Bauersachs and Jochen Bick (ETH Zurich) for their bioinformatics support using Galaxy custom tools. We also would like to thank Dr. Bauersachs for constructive reading and advice about this manuscript.

30

This work was supported by the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills fellowship under grant agreement n° 267196 and the EU project FECUND under the grant agreement nº 312097. This work was also supported by EMMA grant programme from Apis-Gene, France.