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Carmen Almiñana, Emilie Corbin, Guillaume Tsikis, Agostinho S Alcântara-Neto, Valérie Labas, Karine Reynaud, Laurent Galio, Rustem Uzbekov, Anastasiia S Garanina, Xavier Druart, and Pascal Mermillod

Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by in vivo and in vitro oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between in vivo and in vitro oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (P < 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.

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Seok Hee Lee, Hyun Ju Oh, Min Jung Kim, and Byeong Chun Lee

Oviduct cells produce a favorable environment for the development of gametes by generating multiple growth factors. Particularly, in canine species, immature oocytes undergo serial maturation processes in the oviduct, while the other mammals already possess matured oocytes in ovulatory follicles. However, little is known about the potential effect exhibited by the components released from canine oviduct cells (OCs) for modulating the biological function of oocytes. Recently, exosomes are regarded as promising extracellular vesicles because they represent considerable data for molecular cargo. Therefore, we first investigated the effect of canine oviductal exosomes (OC-Exo) on oocyte development via EGFR/MAPK pathway. Our results showed that OC-Exo labeled with PHK67 are successfully incorporated with cumulus cells and oocytes during IVM. Also, OC-Exo markedly increased the proportion of cumulus-oocyte complexes (COCs) exhibiting cumulus expansion as well as cumulus cell proliferation and maturation rate of oocytes (P < 0.05). Furthermore, gene expression patterns related with EGFR/MAPK pathway including EGFR, PKA, TACE/ADAM17, MAPK1/3, MAPK14, PTGS2, TNFAIP6, GDF9, and BMP15 were positively modified in COCs cultured with OC-Exo (P < 0.05). In addition, OC-Exo significantly up-regulated the protein expression levels of p-EGFR, p-MAPK1/3, GDF9 and BMP15 in COCs (P < 0.05). Consequently, the current study provides a model for understanding the roles of OC-Exo as bioactive molecules for canine oocyte maturation via EGFR/MAPK pathway, which would open a new avenue for the application of exosomes to improve assisted reproductive technology in mammals, including humans.

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Eduardo S Ribeiro, José E P Santos, and William W Thatcher

Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants.

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Mahrou Sadri, Jiang Shu, Stephen D Kachman, Juan Cui, and Janos Zempleni

Exosomes facilitate cell-to-cell communication by transferring regulatory molecules such as miRNA from donor to recipient cells, for example, miR-21-5p and miR-30d promote placentation. Exosomes and their miRNA cargos are not exclusively obtained from endogenous synthesis but may also be absorbed from dietary sources, such as milk. This study assessed the effects of milk exosomes and miRNA cargos on embryo development and fertility in C57BL/6 mice. Fluorophore-labeled milk exosomes, miR-21-5p and miR-30d accumulated in murine placenta and embryos following oral gavage. Seventeen mRNAs, miR-21-5p and miR-30d were differentially expressed in placentas of pregnant mice fed a milk exosome and RNA-depleted (ERD) diet or a milk exosome and RNA-sufficient (ERS) diet. Eight of these mRNAs encode proteins implicated in the synthesis of extracellular matrix components, cell adhesion and migration. Changes in mRNA expression were associated with corresponding changes in protein expression, for example, collagen type I. The size of litters born to dams fed ERD was 25–50% smaller than those born to ERS controls. This study implicates dietary exosomes and miRNA in placenta development and embryo survival.

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Mancy Tong, Qi Chen, Joanna L James, Michelle R Wise, Peter R Stone, and Lawrence W Chamley

Throughout human gestation, the placenta extrudes vast quantities of extracellular vesicles (EVs) of different sizes into the maternal circulation. Although multinucleated macro-vesicles are known to become trapped in the maternal lungs and do not enter the peripheral circulation, the maternal organs and cells that smaller placental micro-vesicles interact with in vivo remain unknown. This study aimed to characterise the interaction between placental micro-vesicles and endothelial cells in vitro and to elucidate which organs placental micro-vesicles localise to in vivo. Placental macro- and micro-vesicles were isolated from cultured human first trimester placental explants by sequential centrifugation and exposed to human microvascular endothelial cells for up to 72 h. In vivo, placental macro- and micro-vesicles were administered to both non-pregnant and pregnant CD1 mice, and after two or 30 min or 24 h, organs were imaged on an IVIS Kinetic Imager. Placental EVs rapidly interacted with endothelial cells via phagocytic and clathrin-mediated endocytic processes in vitro, with over 60% of maximal interaction being achieved by 30 min of exposure. In vivo, placental macro-vesicles were localised exclusively to the lungs regardless of time of exposure, whereas micro-vesicles were localised to the lungs, liver and kidneys, with different distribution patterns depending on the length of exposure and whether the mouse was pregnant or not. The fact that placental EVs can rapidly interact with endothelial cells and localise to different organs in vivo supports that different size fractions of placental EVs are likely to have different downstream effects on foeto–maternal communication.

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Irene Ruiz-González, Jing Xu, Xiaoqiu Wang, Robert C Burghardt, Kathrin A Dunlap, and Fuller W Bazer

Conceptus–endometrial communication during the peri-implantation period of pregnancy ensures establishment of pregnancy. We hypothesized that this dialog involves exosomes, ovine endogenous jaagsiekte retroviruses (enJSRV) and toll-like receptors (TLR) which regulate the secretion of interferon tau (IFNT), the pregnancy recognition signal in ruminants. First, exosomes isolated from uterine flushings from cyclic and pregnant ewes were analyzed for exosomal content and uterine expression of heat shock protein 70 (HSC70). Then, conceptus trophectoderm cells (oTr1) treated with different doses of exosomes were analyzed for the expression of genes involved in TLR-mediated cell signaling. The results revealed that exosomes contain mRNAs for enJSRV-ENV, HSC70, interleukins, and interferon (IFN)-regulatory factors. Exosomal content of enJSRV-ENV mRNA and protein decreased from days 10 and 12 to day 16 of gestation, and uterine expression of HSC70 increased in pregnant ewes compared with cyclic ewes. The oTr1 cells proliferated and secreted IFNT in a dose-dependent manner in response to exosomes from cyclic ewes. The expression of CD14, CD68, IRAK1, TRAF6, IRF6, and IRF7 mRNAs that are key to TLR-mediated expression of type 1 IFNs was significantly influenced by day of pregnancy. This study demonstrated that exosomes are liberated into the uterine lumen during the estrous cycle and early pregnancy; however, in pregnant ewes, exosomes stimulate trophectoderm cells to proliferate and secrete IFNT coordinately with regulation of TLR-mediated cell signaling. These results support our hypothesis that free and/or exosomal enJSRV act on the trophectoderm via TLR to induce the secretion of IFNT in a manner similar to that for innate immune responses of macrophages and plasmacytoid dendritic cells to viral pathogens.

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Daqian Dong, Jinmeng Yang, Yining Chen, Guofan Peng, Heran Cao, Huihui Gao, Tianqi Jin, Fangxia Yang, and Wuzi Dong

Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

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Inga Laezer, Sergio E Palma-Vera, Fan Liu, Marcus Frank, Nares Trakooljul, Andreas Vernunft, Jennifer Schoen, and Shuai Chen

In mammals, around the time of ovulation, the hormonal profile dynamically changes in synchrony with reproductive events occurring in the oviduct, that is, sperm arrival, fertilization, and early embryo development. Extracellular vesicles (EVs) have been recently recognized as key components of the embryonic milieu; however, composition and function of oviductal EVs during this crucial period remains to be further explored. Therefore, we initially characterized EVs from porcine oviductal fluid specifically around the critical ovulation window: that is, estrus (E), late estrus (LE, day of expected ovulation), post ovulation (PO), and additionally diestrus (D). Total EV numbers gradually rose from D to E, LE and PO (P < 0.05), which corresponded to the total EV protein amount (P < 0.05). Strikingly, the mean size of EVs in PO was significantly smaller than in E and LE groups, which also had a lesser proportion of small EVs (P < 0.05). The EV protein cargoes during the periovulatory period were further analyzed by mass spectrometry. Qualitative analysis detected 1118 common proteins, which are most enriched in the cellular component of EVs/exosomes. Hierarchical clustering indicated similar protein profile within the biological replicates, but large discrepancy among stages. Further quantitative analysis discovered 34 and 4 differentially expressed proteins in the comparison between E and PO and in the comparison between E and LE, respectively. The dynamic EV protein profile together with the quick adaption in EV size and quantity suggests that porcine oviductal EV secretion are under the hormonal influence during the estrus cycle.

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Virginie Barraud-Lange, Céline Chalas Boissonnas, Catherine Serres, Jana Auer, Alain Schmitt, Brigitte Lefèvre, Jean-Philippe Wolf, and Ahmed Ziyyat

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

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Meritxell Jodar

The paternal contribution to the new individual is not just limited to half the diploid genome. Recent findings have shown that sperm delivers to the oocyte several components, including a complex population of RNAs, which may influence early embryo development and the long-term phenotype of the offspring. Although the majority of sperm RNAs may only represent spermatogenic leftovers with no further function, the male gamete provides a specific set of RNAs to the oocyte that is able to modulate gene expression in the preimplantation embryo. Those sperm transcripts include coding and non-coding RNAs that might either be translated by the oocyte machinery or directly regulate embryo gene expression at the transcriptional or post-transcriptional level. Interestingly, some sperm RNAs seem to be acquired during post-testicular maturation through active communication between sperm and epididymal and seminal exosomes released by the epididymis and the male accessory sex glands, respectively. Exosomes contained in the seminal plasma seem to not only interact with the spermatozoa but also with cells from the female reproductive tract, modulating their gene expression and influencing female immune response triggered by the semen. This review also considers the findings that indicate the role of semen RNAs in preimplantation embryo development and offspring phenotypes. In this regard, different studies supporting the hypothesis of paternal epigenetic inheritance of altered metabolic phenotypes associated with environmental exposures are discussed. Lastly, potential mechanisms that could explain the impact of semen RNAs to both early embryogenesis and paternal epigenetic inheritance are suggested.