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In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction that selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation, and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner., After mating or insemination, the region-specific and hormonally regulated morphology and secretions of the utero-tubal junction and oviduct lead to the selection of a limited sub-population of top quality spermatozoa at the site of fertilization.

International audience; Follicular fluid (FF) fills the interior portion of the ovarian antral follicle and provides a suitable microenvironment for the growth of the enclosed oocyte through molecular factors that originate from plasma and the secretions of follicular cells. FF contains extracellular nanovesicles (ffEVs), including 30–100-nm membrane-coated exosomes, which carry different types of RNA, proteins, and lipids and directly influence oocyte competence to develop embryo. In the present study, we aimed to characterize the protein cargo of EVs from the FF of 3–6-mm follicles and uncover the origins of ffEVs by assessing expression levels of corresponding mRNAs in bovine follicular cells and oocyte and cell proteomes. Isolated exosome-like ffEVs were 53.6 + 23.3 nm in size and could be internalized by cumulus-oocyte complex. Proteomes of ffEVs and granulosa cells (GC) were assessed using nanoflow liquid chromatography coupled with high-resolution tandem mass spectrometry after the gel fractionation of total proteins. In total, 460 protein isoforms corresponding to 322 unique proteins were identified in ffEVs; among them, 190 were also identified via GC. Gene Ontology terms related to the ribosome, protein and RNA folding, molecular transport, endocytosis, signal transduction, complement and coagulation cascades, apoptosis, and developmental biology pathways, including PI3K-Akt signaling, were significantly enriched features of ffEV proteins. FfEVs contain numerous ribosome and RNA-binding proteins, which may serve to compact different RNAs to regulate gene expression and RNA degradation, and might transfer ribosomal constituents to the oocyte. Majority of genes encoding ffEV proteins expressed at different levels in follicular cells and oocyte, corroborating with numerous proteins, which were reported in bovine oocyte and cumulus cells in other studies thus indicating possible origin of ffEV proteins. The limited abundance of several mRNAs within follicular cells indicated that corresponding ffEV proteins likely originated from circulating exosomes released by other tissues. Analysis of bovine ffEV transcriptome revealed that mRNAs present in ffEV accounted for only 18.3% of detected ffEV proteins. In conclusion, our study revealed numerous proteins within ffEVs, which originated from follicular and other cells. These proteins are likely involved in the maintenance of follicular homeostasis and may affect oocyte competence.

International audience; Sperm migration through the female genital tract is not a quiet journey. Uterine contractions quickly operate a drastic selection, leading to a very restrictive number of sperm reaching the top of uterine horns and finally, provided the presence of key molecules on sperm, the oviduct, where fertilization takes place. During hours and sometimes days before fertilization, subpopulations of spermatozoa interact with dynamic and region-specific maternal components, including soluble proteins, extracellular vesicles and epithelial cells lining the lumen of the female tract. Interactions with uterine and oviductal cells play important roles for sperm survival as they modulate the maternal immune response and allow a transient storage before ovulation. The body of work reported here highlights the importance of sperm interactions with proteins originated from both the uterine and oviductal fluids, as well as hormonal signals around the time of ovulation for sperm acquisition of fertilizing competence.

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4&ndash, 5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4&ndash, 6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

International audience; High polyspermy is one of the major limitations of porcine in vitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2 mg mL(-1)), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30-150 nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve in vitro embryo production outcomes.

International audience; Infertility is a worldwide problem affecting around 48.5 million couples in the word, the male factor being responsible for approximately the 50% of the cases, with a high percentage of unknown causes. For that reason, improving the success of In Vitro Fertilization (IVF) techniques is a primordial aim for researchers working in the reproductive field. Here, by using a mammalian animal model, the bovine, we present an innovative in vitro fertilization system that combines the use of a somatic component, the epithelial oviductal cells, and a carbon-based material, the graphene oxide, with the aim to open new ways in IVF systems design and application. Our results show an increase in the IVF outcomes without harming the blastocyst developmental rate, as well as high modified proteomic and lipidomic profiles of capacitating spermatozoa. Furthermore, we compared the modifications produced by GO with those exerted by the hormone progesterone, finding similar functional effects on sperm capacitation. In conclusion, our results stand out the use of a non-physiological material as graphene oxide in a new and innovative strategy that improves sperm capacitation, conferring them a higher fertilizing competence and thus increasing the in vitro fertilization outcomes. (C) 2019 Published by Elsevier Ltd.

Background The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. Results We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. Conclusions Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success. Electronic supplementary material The online version of this article (10.1186/s12864-018-4982-5) contains supplementary material, which is available to authorized users.

Although essential for artificial insemination (AI) and MOET (multiple ovulation and embryo transfer), oestrus synchronisation and superovulation are associated with increased female reproductive tract mucus production and altered sperm transport. The effects of such breeding practices on the ovine cervicovaginal (CV) mucus proteome have not been detailed. The aim of this study was to qualitatively and quantitatively investigate the Merino CV mucus proteome in naturally cycling (NAT) ewes at oestrus and mid-luteal phase, and quantitatively compare CV oestrus mucus proteomes of NAT, progesterone synchronised (P4) and superovulated (SOV) ewes. Quantitative analysis revealed 60 proteins were more abundant during oestrus and 127 were more abundant during the luteal phase, with 27 oestrus specific and 40 luteal specific proteins identified. The oestrus proteins most disparate in abundance compared to mid-luteal phase were ceruloplasmin (CP), chitinase-3-like protein 1 (CHI3L1), clusterin (CLU), alkaline phosphatase (ALPL) and mucin-16 (MUC16). Exogenous hormones greatly altered the proteome with 51 and 32 proteins more abundant and 98 and 53 proteins less abundant, in P4 and SOV mucus, respectively when compared to NAT mucus. Investigation of the impact of these proteomic changes on sperm motility and longevity within mucus may help improve sperm transport and fertility following cervical AI. Significance This manuscript is the first to detail the proteome of ovine cervicovaginal mucus using qualitative and quantitative proteomic methods over the oestrous cycle in naturally cycling ewes, and also after application of common oestrus synchronisation and superovulation practices. The investigation of the mucus proteome throughout both the follicular and luteal periods of the oestrous cycle, and also after oestrous synchronisation and superovulation provides information about the endocrine control and the effects that exogenous hormones have on protein expression in the female reproductive tract. This information contributes to the field by providing important information on the changes that occur to the cervicovaginal mucus proteome after use of exogenous hormones in controlled breeding programs, which are commonly used on farm and also in a research setting.

Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC–MS/MS and shotgun proteomic analysis by 2D–LC–MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. Biological significance This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography-tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.

Controlled breeding programmes utilising exogenous hormones are common in the Australian sheep industry, however the effects of such programmes on cervicovaginal mucus properties are lacking. As such, the aim of this study was to investigate cervicovaginal (CV) mucus from naturally cycling (NAT), progesterone synchronised (P4), prostaglandin synchronised (PGF2α), and superovulated (SOV) Merino ewes. Experiment 1; volume, colour, spinnbarkeit, chemical profile and protein concentration of mucus (NAT, P4, PGF2α and SOV; n = 5 ewes/treatment) during the follicular (5 d) and luteal phases (8 d) was investigated. Experiment 2; in vivo mucus pH and in vitro mucus penetration by frozen-thawed spermatozoa (NAT, P4 and SOV; n = 11 ewes/treatment) was investigated over oestrus (2 d) and the mid-luteal phase (pH only, 2 d). Oestrus mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus (p < 0.05). SOV increased mucus production and protein concentration (p < 0.05) while PGF2α reduced mucus volume (p < 0.05). Mucus pH (oestrus 6.2–6.5), chemical profile and mucus penetration by sperm were unchanged (p > 0.05). Results indicate that exogenous hormones used for controlled breeding affect cervicovaginal mucus production, but few other tested characteristics. Further research is required to explain fertility differences between synchronised and naturally cycling animals following cervical AI.

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.

Remerciements : Aurélien Brionne, INRA, Unité des Recherches Avicoles, Centre Tours Val de Loire; Our objective was to investigate the regulation of the proteome in the bovine oviductal fluid according to the stage of the oestrous cycle, to the side relative to ovulation and to local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected at four stages of the oestrous cycle: pre-ovulatory (Pre-ov), post-ovulatory (Post-ov), mid- and late luteal phases from adult cyclic cows (18-25 cows/stage). The proteomes were assessed by nanoLC-MS/MS and quantified by label free method. A total of 482 proteins were identified including a limited number specific to one stage or one side. Proportions of differentially abundant proteins fluctuated from 10 to 24% between sides at one stage and from 4 to 20% among stages in a given side of ovulation. In oviductal fluids ipsilateral to ovulation, Annexin A1 was the most abundant protein at Pre-ov compared to Post-ov while numerous heat shock proteins were more abundant at Post-ov compared to Pre-ov. Among differentially abundant proteins, seven tended to be correlated with intra-oviductal concentrations of progesterone. A wide range of biological processes was evidenced for differentially abundant proteins, among which metabolic and cellular processes were predominant. This work identifies numerous new candidate proteins potentially interacting with the oocyte, spermatozoa and embryo to modulate fertilization and early embryo development.

STUDY HYPOTHESIS Is the c-Jun-N-terminal kinase (JNK) pathway implicated in primordial follicle activation? STUDY FINDING Culture of ovine ovarian cortex in the presence of two different c-Jun phosphorylation inhibitors impeded pre-antral follicle activation. WHAT IS KNOWN ALREADY Despite its importance for fertility preservation therapies, the mechanisms of primordial follicle activation are poorly understood. Amongst different signalling pathways potentially involved, the JNK pathway has been previously shown to be essential for cell cycle progression and pre-antral follicle development in mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovine ovarian cortex pieces were cultured with varying concentrations of SP600125, JNK inhibitor VIII or anti-Mullerian hormone (AMH) in the presence of FSH for 9 days. Follicular morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptosis and follicle activation (Foxo3a) were assessed. MAIN RESULTS AND THE ROLE OF CHANCE Inhibition of primordial follicle activation occurred in the presence of SP600125, JNK inhibitor VIII and AMH when compared with controls (all P < 0.05) after 2 days of culture. However, only in the highest concentrations used was the inhibition of activation associated with induction of follicular apoptosis (P < 0.05). In growing follicles, PCNA antigen expression was reduced when the JNK inhibitors or AMH were used (P < 0.05 versus control), indicating reduced proliferation of the somatic compartment. LIMITATIONS, REASONS FOR CAUTION Although we evaluated the effects of inhibition of c-Jun phosphorylation on primordial follicle development, we did not determine the cellular targets and mechanism of action of the inhibitors. WIDER IMPLICATIONS OF THE FINDINGS These results are the first to implicate the JNK pathway in primordial follicle activation and could have significant consequences for the successful development of fertility preservation strategies and our understanding of primordial follicle activation. LARGE SCALE DATA n/a. STUDY FUNDING AND COMPETING INTERESTS Dr Michael J. Bertoldo and the laboratories involved in the present study were supported by a grant from 'Region Centre' (CRYOVAIRE, Grant number #320000268). There are no conflicts of interest to declare.

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC–MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24 h in milk at 15 °C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24 h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. Biological significance Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24 h preservation period.

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10μM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–100 nm) membrane-bound vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system in the uterus. However, little is known about the role of these vesicles in the oviduct. Our study aimed at (1) demonstrating the existence of oviducal-embryo communication via exosomes, (2) deciphering the exosomal dialogue between them at the proteomic level, and (3) comparing the exosomal proteomic content to the oviducal fluid proteomic content in order to highlight the key role of exosomes in this dialogue. Cow oviducts (pool of 6 oviducts at different stages of the cycle in 4 replicates) were flushed, and exosomes were isolated by serial ultracentrifugation. Exosomes were measured by dynamic light scattering analysis, resulting in exosomes (63.25–97.03 nm) and microvesicle observations (>100 nm). Bovine embryos were produced in vitro up to the blastocyst and hatching/hatched blastocyst stages. To demonstrate the existence of the oviducal-embryo communication via exosomes, oviducal exosomes were labelled with green fluorescent dye (PKH67), filtered (0.22 µm) to remove microvesicles, and co-incubated with blastocysts and hatching/hatched (H) blastocysts for 20 h, under 5% CO2 and 5% O2 conditions. Subsequently, embryos were washed in exosome-free medium, fixed in 4% paraformaldehyde, and labelled with Hoechst 33342 and Actin Red Phallodin. Confocal microscopy observations confirmed that exosomes were internalized by blastocysts and H-blastocysts and located around the nucleus, demonstrating the existence of an oviducal-embryo communication via exosomes. Moreover, our results showed that the zona pellucida does not represent a barrier for exosomes and they act as natural nanoshuttles bringing oviducal signals into the embryo. Then, proteomic analysis by LC1D-nanoESI-LTQ-Orbitrap was used to decipher oviducal exosomal content, identifying 480 proteins. Gene ontology analysis revealed that a high number of these proteins were involved in metabolism (24.9%), cellular process (19.3%), and 0.8% reproductive processes. Further analysis revealed that more than 56% of exosomal proteins involved in cellular process were associated with cell-to-cell communication. Finally, exosomal proteins were compared with proteins present in oviducal fluid from a pool of samples from cows at Day 0 and Day 10 of the oestrous cycle. Comparative analysis showed that from a total of 607 proteins identified in both oviducal exosomes and fluid sources, 105 were specific to exosomes, 127 were specific to fluid, whereas 375 were common to both sources. Our findings provide the first evidence of oviducal-embryo communication via exosomes, an important first step in furthering the understanding of the oviducal environment and the role of exosomes as early mediators of embryo-maternal cross talk. This research was supported by the EU AgreenSkills fellowship n° 267196 and EU FECUND Project no 312097.

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–200 nm) membrane vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system. Exosomes have been identified in vivo in all body fluids including follicular, uterine, and oviductal fluids and can be secreted by most cell types in vitro. Bovine oviductal epithelial cells (BOEC) have been thoroughly used to study embryo-maternal communication and to improve embryo development in vitro. Hence, our objective was to provide a morphologic and proteomic characterisation of exosomes secreted by BOEC in vivo in the oviductal fluid and in vitro in the conditioned media. Oviducts from cows were flushed to recover in vivo exosomes and then BOEC were scraped in order to derive primary cultures. In vitro exosomes were collected from conditioned media of BOEC primary cultures after reaching confluence (10 days). Isolation of exosomes from in vivo and in vitro origin was performed by ultracentrifugation. The presence of exosomes was confirmed in oviductal flushings and conditioned media by electron microscopy. Further characterisation of exosomes was carried out based on morphology (transmission electron microscopy), size (dynamic light scattering, DLS), and protein composition (protein profile analysis by SDS-PAGE and Western immunoblotting). Preliminary results by DLS revealed different size distribution profiles in exosome samples (in vivo: mean size of 93.41 nm; in vitro: 433.5 nm). Because exosomes are considered as “micromaps” of the originating cells, protein patterns expressed by in vivo exosomes and in vitro exosomes were compared with scraped and cultured BOEC, respectively. Protein profile analysis by SDS-PAGE showed quantitative and qualitative differences among the exosome samples, their cells of origin, and the milieu (conditioned media or flushing). Exosome-specific protein bands were detected and will be further characterised. In addition, exosomes from in vivo and in vitro origin exhibited distinct proteomic profiles. Western blot analysis demonstrated that (1) both exosomal protein samples were positive for HSP70, a known exosomal protein, and negative for Grp78, an endoplasmic reticulum marker detected in BOEC; (2) in vivo exosomes expressed oviductal glycoprotein (OVGP), heat shock protein A8 (HSPA8), and myosin 9 (MYH9), 3 oviductal proteins with known roles in fertilization and early pregnancy. However, only HSPA8 and MYH9 were detected in in vitro exosomes. Our results provide the first extensive characterisation of oviductal exosomes from in vivo and in vitro origin, an essential step in furthering our understanding of the early embryo-maternal cross talk.