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Preterm labor (PTL) and Preterm Premature Rupture of Membranes (PPROM) impose substantial morbimortality on mothers and newborns. Exosomes act in intercellular communication carrying molecules involved in physiopathological processes. Little is known about exosomal proteins in prematurity. Our aim was to evaluate the protein expression of hemopexin, C1 inhibitor (C1INH) and alpha-2-macroglobulin (A2M) from circulating exosomes of women with PTL and PPROM. Plasma was obtained from PTL, PPROM, Term in labor and Term out of labor (T) patients, exosomes were isolated by ultracentrifugation, then lysed and the proteins quantified. Western Blot (WB) and Nanoparticle Tracking Analysis (NTA) were performed. Data were compared by Kruskal-Wallis, unpaired T-test and one-way ANOVA. WB and NTA confirmed exosome isolation (concentration: 4.3 × 10 10  particles/ml ± 1.9 × 10 10 ). There was no difference regarding hemopexin or C1INH expression between the groups. For A2M, the fold change was significantly higher on preterm groups when compared to term groups (1.07 ± 0.30 vs. 0.42 ± 0.17, p < 0.0001). Higher levels of A2M in circulating exosomes are linked to preterm pregnancies. sEV are strong candidates to intermediate maternal-fetal communication, carrying preterm labor-related immunomodulatory proteins.

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to complex interaction between the host immune response and the infecting virus. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes carrying viral proteins or inflammatory molecules, which can participate in the chronic inflammation that is observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line, evaluated for the tax mRNA presence, and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokines, IFN-γ and TNF-α, were upregulated in T cells after treatment of the PBMC with Tax-carrying exosomes compared to the negative control. Interleukin-4, Granzyme B, and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying tax -mRNA isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th) 1 , and not Th 2 -immune response. If this finding is further confirmed, this study may have impact on investigations on the pathogenesis of HAM-TSP and the inflammatory response involved in this disease.

Mammalian gamete maturation requires extensive signaling between germ cells and their surrounding somatic cells. In the ovary, theca cells, mural granulosa cells, cumulus cells and the oocyte all secrete factors throughout follicle growth and maturation that are critical for ovulation of a high-quality oocyte with the competence to develop into an embryo. Similarly, maturation of sperm occurs as it transits the epididymis during which epididymal epithelium and sperm exchange secretory factors that are required for sperm to gain motility and fertility. Recent studies in a variety of species have uncovered the presence of cell-secreted vesicles in follicular fluid (microvesicles and exosomes) and epididymal fluid (epididymosomes). Moreover, these cell-secreted vesicles contain small non-coding regulatory RNAs called microRNAs, which can be shuttled between maturing gametes and surrounding somatic cells. Although little is known about the exact mechanism of how microRNAs are loaded into these cell-secreted vesicles or are transferred and modulate gene expression and function in gametes, recent studies clearly suggest that cell-secreted vesicle microRNAs play a role in oocyte and sperm maturation. Moreover, a role for cell-secreted vesicular microRNAs in gamete maturation provides for novel opportunities to modulate and discover new diagnostic markers associated with male or female fertility. This manuscript provides an overview of cell-secreted vesicles in ovarian follicular fluid and epididymal fluid and microRNAs and discusses recent discoveries on the potential function of cell-secreted vesicles as carriers of microRNAs in oocyte and sperm maturation., (© 2018 Society for Endocrinology.)

Nuclear reprogramming mediated by somatic cell nuclear transfer (SCNT) has many applications in medicine. However, animal clones show increased rates of abortion and reduced neonatal viability. Herein, we used exosomal-miRNA profiles as a non-invasive biomarker to identify pathological pregnancies. MiRNAs play important roles in cellular proliferation and differentiation during early mammalian development. Thus, the aim of this study was to identify exosomal-miRNAs in maternal blood at 21 days of gestation that could be used for diagnosis and prognosis during early clone pregnancies in cattle. Out of 40 bovine-specific miRNAs, 27 (67.5%) were with low abundance in the C-EPL (Clone - Early pregnancy loss) group compared with the C-LTP (Clone - Late pregnancy) and AI-LTP (Artificial Insemination - Late pregnancy) groups, which had similar miRNAs levels. Bioinformatics analysis of the predicted target genes demonstrated signaling pathways and functional annotation clusters associated with critical biological processes including cell proliferation, differentiation, apoptosis, angiogenesis and embryonic development. In conclusion, our results demonstrate decreased exosomal-miRNAs in maternal blood at 21 days of gestation in cloned cattle pregnancies that failed to reach term. Furthermore, the predicted target genes regulated by these 27 miRNAs are strongly associated with pregnancy establishment and in utero embryonic development.

The ovarian follicular fluid (FF) is a complex fluid that constitutes the microenvironment of developing follicles and contains factors secreted by the surrounding cells and blood plasma compounds that cross the "blood-follicle barrier." Upon oocyte retrieval (in human, bovine, and equine) the follicular fluid is normally discarded and represents a repertoire of cellular messages exchanged during follicle development, thus providing a suitable sample for performing oocyte quality diagnostics. Several studies report on the presence of extracellular vesicles (EVs) in FF from human, bovine and equine. Here, we describe the process of FF collection from human and bovine and the enrichment and isolation of EVs that we termed folliculosomes (FFEs), using available commercial kits as well as the traditional ultracentrifugation methods.

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy., (© 2016 by the Society for the Study of Reproduction, Inc.)

Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Proliferation and Organ Development pathways, while umbilical serum exosomal and placentomes miRNAs were predicted to target cellular development and organismal/embryonic development., (© 2014 Wiley Periodicals, Inc.)

Background: Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50-150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation., Methods: Follicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30-34 h after induction of follicular maturation). A real time PCR screen was conducted to reveal significant differences in the presence of exosomal miRNAs isolated from mid-estrous and pre-ovulatory follicles, and according to bioinformatics analysis these exosomal miRNAs are predicted to target members belonging to the TGFB superfamily, including ACVR1 and ID2. Granulosa cells from pre-ovulatory follicles were cultured and treated with exosomes isolated from follicular fluid. Changes in mRNA and protein were measured by real time PCR and Western blot., Results: ACVR1 mRNA and protein was detected in granulosa cells at mid-estrous and pre-ovulatory stages, and real time PCR analysis revealed significantly lower levels of ID2 (an ACVR1 target gene) in granulosa cells from pre-ovulatory follicles. Exposure to exosomes from follicular fluid of mid-estrous follicles decreased ID2 levels in granulosa cells. Moreover, exosomes isolated from mid-estrous and pre-ovulatory follicles contain ACVR1 and miR-27b, miR-372, and miR-382 (predicted regulators of ACVR1 and ID2) were capable of altering ID2 levels in pre-ovulatory granulosa cells., Conclusions: These data indicate that exosomes isolated from follicular fluid can regulate members of the TGFB/BMP signaling pathway in granulosa cells, and possibly play a role in regulating follicle maturation.

Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.