Search

Unraveling the oviduct fluid (OF) and its regulation is crucial to understand the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs. contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs. post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n=32 OF pools for all region ´ stage ´ side conditions). A total of 3,760 proteins were identified in the OF, of which 37% were predicted to be secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo.

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; 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 control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (β-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca β-NGF (aβ-NGF) and its 3D structure and compared it with human recombinant β-NGF (hβ-NGF). We showed that the β-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that β-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

International audience; The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) - 3 and - 5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

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.

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs&rsquo, metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development.

International audience; Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

International audience; During ejaculation and deposition in the female genital tract, spermatozoa are exposed to seminal plasma, a mix of secretions primarily from the accessory sex glands. Proteins, which make up the largest contribution to seminal plasma by weight, have been the focus of much interest, in particular the identification of specific proteins both in the plasma and/or found bound to the sperm surface post ejaculation. Global proteomic studies of seminal plasma originating from a range of species over the last 15 years have revealed their hitherto unknown diversity and complexity. Seminal plasma is generally known to aid sperm survival and fertility in a range of species and studies have begun to reveal its link with sperm function and identification, as markers of fertility. This review summarises recent data on proteins found on the sperm surface that originate from seminal plasma and have subsequently been shown to correlate with fertility, with a focus on the pig. (C) 2019 Published by Elsevier Inc.

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.

International audience; During ejaculation and the deposition in the female genital tract, spermatozoa undergo hypo-osmotic stress and need to withstand it for optimal fertility. Resistance to hypo-osmotic stress may be affected by the interaction of the spermatozoa with seminal fluid components. The hypo-osmotic resistance of epididymal and ejaculated spermatozoa from dogs, rams and boars was assessed by flow cytometric measurement of sperm viability after incubation in NaCl solutions with osmolalities ranging from 0 to 300 mmol/kg. The hypotonic resistance of epididymal spermatozoa was greater than those of ejaculated spermatozoa in all three species. Among species comparison revealed that ejaculated spermatozoa from dogs were much more resistant than those from rams and boars as 80.4 ± 5.3%, 56.7 ± 4.7 and 9.6 ± 3.6% of live spermatozoa were observed following exposure to an osmolality of 90 mmol/kg in dogs, rams and boars respectively. This can be explained by the fact that dog, ram and boar differ markedly in composition of the seminal plasma owing to the presence (ram, boar) or absence (dog) of seminal vesicles. Hypotonic resistance of epididymal and ejaculated dog spermatozoa was similar whereas ram and boar spermatozoa showed a marked drop in resistance after ejaculation. The in vitro incubation of boar epididymal spermatozoa with raw seminal plasma or the seminal plasma protein fraction induced a similar loss of resistance, suggesting that seminal proteins are involved in the lack of resistance to hypotonic stress of boar ejaculated spermatozoa.

The interactions between oviductal fluid (OF) proteins and spermatozoa play major roles in sperm selection, storage and capacitation before fertilization. However, only a few sperm-interacting proteins in the OF has been identified and very little is known about the regulation of sperm-oviduct interactions across the estrous cycle. Samples of bovine frozen-thawed sperm from three bulls were incubated with OF at pre-, post-ovulatory stages (Pre-/Post-ov) or luteal phase (LP) of the estrous cycle (7 mg/mL proteins, treated groups) or with a protein-free media (control). The proteomes of sperm cells were assessed by nanoLC–MS/MS and quantified by label-free methods. A total of 27 sperm-interacting proteins originating in the OF were identified. Among those, 14 were detected at all stages, eight at Post-ov and LP and five only at LP. The sperm-interacting proteins detected at all stages or at LP and Post-ov were on average more abundant at LP than at other stages (P

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.

National audience

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.

International audience; The source and composition of seminal plasma has previously been shown to alter the ability of spermatozoa to survive cryopreservation. In the present study, the ionic and proteomic composition of seminal plasma from rams with high (HSP; n = 3) or low (LSP; n = 3) freezing resilient spermatozoa was assessed. 75 proteins were identified to be more abundant in HSP and 48 proteins were identified to be more abundant in LSP. Individual seminal plasma proteomes were established for each of the six rams examined. For each ram, correlations were conducted between previously recorded freezing resilience [1] and individual spectral counts in order to identify markers of freezing resilience. 26S proteasome complex, acylamino acid releasing enzyme, alpha mannosidase class 2C, heat shock protein 90, tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase and transitional endoplasmic reticulum ATPase were found to be positively correlated (r2 > 0.7) with freezing resilience. Cystatin, zinc-2-alpha glycoprotein, angiogenin-2-like protein, cartilage acidic protein-1, cathepsin B and ribonuclease 4 isoform 1 were found to be negatively correlated (r2 > 0.7) with freezing resilience. Several negative markers were found to originate from the accessory sex glands, whereas many positive markers originated from spermatozoa and were part of or associated with the 26S proteasome or CCT complex.

The fertility after use of liquid stored ram semen following cervical AI rapidly decreases if semen is stored beyond 12 h. The dilution of seminal plasma is often cited as a key contributor to the diminished motility and fertility of ram spermatozoa subjected to liquid preservation. Two experiments were conducted to assess the effect of spermatozoa concentration (i.e. dilution rate) and percentage of seminal plasma on the motility and viability of liquid stored ram spermatozoa. In Experiment 1, semen was diluted to one of seven concentrations ranging from 0.2 to 1.4 × 109 spermatozoa/ml with milk and assessed for motility after 3 or 24 h of storage at 15 °C. In Experiment 2, semen was collected and washed to remove seminal plasma before re-dilution to 0.2–1.4 × 109 spermatozoa/ml with milk containing 0%, 20% or 40% (final v/v ratio) seminal plasma and assessed for viability and motility after 3 or 24 h of storage at 15 °C. Whereas motility was not affected by spermatozoa concentration after 3 h of storage, the proportion of progressive spermatozoa decreased after 24 h of storage when spermatozoa concentration was greater than 1.0 × 109 spermatozoa/ml. The duration of preservation and the spermatozoa concentration affected spermatozoa motility but had no impact on spermatozoa viability. This negative effect of greater spermatozoa concentrations on motility was independent of the presence and the concentration of seminal plasma. The seminal plasma at both concentrations (20% and 40%) had a protective effect on spermatozoa motility after 24 h of storage. These findings have the potential to improve the efficiency of cervical AI with liquid stored ram semen.

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.

National audience