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. 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 < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. 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.

The oviduct is the anatomical part of the female reproductive tract where the early reproductive events take place, from gamete transport, fertilization and early embryo development to the delivery of a competent embryo to the uterus, which can implant and develop to term. The success of all these events rely upon a two-way dialogue between the oviduct (lining epithelium and secretions) and the gametes/embryo(s). Recently, extracellular vesicles (EVs) have been identified as major components of oviductal secretions and pointed to as mediators of the gamete/embryo-maternal interactions. EVs, comprising exosomes and microvesicles, have emerged as important agents of cell-to-cell communication by the transfer of biomolecules (i.e., mRNAs, miRNAs, proteins) that can modulate the activities of recipient cells. Here, we provide the current knowledge of EVs in the oviductal environment, from isolation to characterization, and a description of the EVs molecular content and associated functional aspects in different species. The potential role of oviductal EVs (oEVs) as modulators of gamete/embryo-oviduct interactions and their implications in the success of early reproductive events is addressed. Lastly, we discuss current challenges and future directions towards the potential application of oEVs as therapeutic vectors to improve pregnancy disorders, infertility problems and increase the success of assisted reproductive technologies.

Extracellular vesicles (EVs) have emerged as novel cell-to-cell communication mediators in physiological and pathological scenarios. Their ability to transfer their molecular cargo (RNAs, proteins and lipids) from one cell to another, in the vicinity or far from the cell of origin, together with their capacity of exerting a functional impact on the target cell make them valuable diagnostic tools as well as therapeutic vectors in a variety of diseases. In the reproductive field, there is a growing interest in the role of EVs in gamete/embryo-maternal communication and their potential implications in the reproductive success. In this review, we provide current knowledge of EVs secreted by the oviduct (oEVs) and embryos (eEVs), since both have been proposed as key players in the crucial two-way dialogue between the oviduct (lining epithelium and secretions) and the embryo that ensures successful pregnancy. Both oEVs and eEVs molecular cargos and their potential role as multi-signal messengers in the gametes/embryo-oviduct cross-talk and in the embryo-to-embryo communication in different species are also addressed. Eventually, a comparative analysis between oEVs and eEVs has been performed to shed some light on common and specific cargos responsible for their functions supporting the early reproductive events and as prime candidate molecules for improving fertility and assisted reproductive technologies outcomes.

Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo–oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by low-input RNA sequencing. Analysis of RNA-seq data revealed 221 differentially expressed genes (DEGs) between FoEV treatment and control, 67 DEGs for FeEV and FoEV treatments, and minor differences between FeEV treatment and control (28 DEGs). An integrative analysis of mRNAs and miRNAs contained in oEVs obtained in a previous study with embryonic mRNA alterations pointed to direct effects of oEV cargo on embryos (1) by increasing the concentration of delivered transcripts; (2) by translating delivered mRNAs to proteins that regulate embryonic gene expression; and (3) by oEV-derived miRNAs which downregulate embryonic mRNAs or modify gene expression in other ways. Our study provided the first high-throughput analysis of the embryonic transcriptome regulated by oEVs, increasing our knowledge on the impact of oEVs on the embryo and revealing the oEV RNA components that potentially regulate embryonic development.

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’ 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.