Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >10 5-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of illdefined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment.

This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T-AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T-AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T-AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.

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Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV-and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant noncoding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EVassociated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.

ABSTRACT:Heat shock protein 70 (HSP70) is considered as a gene which affects semen quality traits. The present study attempted to investigate the relationship between the HSP70 expression level and motility of bull sperm during the process of freezing-thawing. Semen samples were collected from 5 QinChuan bulls by artificial vagina. Sperm motility and plasma membrane integrity of the semen samples at three stages (fresh, after equilibration, and frozen-thawed) were evaluated. The HSP70 expression level at the three stages was detected using real-time PCR. The results indicated that HSP70 expression level, membrane integrity, and sperm motility in the fresh semen were higher than those of the sperm after equilibration and freezing-thawing (P < 0.05), the HSP70 expression level, plasma membrane integrity, and sperm motility in sperm after equilibration were higher than those of the frozenthawed sperm (P < 0.05). The correlation between HSP70 expression level and sperm motility was positive (ranging from 0.327 to 0.785). The results suggest that HSP70 expression level in bull spermatozoa was gradually decreased following the process of freezing-thawing, and might be associated with bull sperm motility.

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This study was conducted to investigate the influence of superoxide dismutase (SOD) on the quality of boar semen during liquid preservation at 17°C. Semen samples from 10 Duroc boars were collected and pooled, divided into five equal parts and diluted with Modena containing different concentrations (0, 100, 200, 300 and 400 U/mL) of SOD. During the process of liquid preservation at 17°C, sperm motility, acrosome integrity, membrane integrity, total antioxidant capacity (T-AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H O ) content were measured and analyzed every 24 h. Meanwhile, effective survival time of boar semen during preservation was evaluated and analyzed. The results indicated that different concentrations of SOD in Modena showed different protective effects on boar sperm quality. Modena supplemented with SOD decreased the effects on reactive oxygen species on boar sperm quality during liquid preservation compared with that of the control group. The added 200 U/mL SOD group showed higher sperm motility, membrane integrity, acrosome integrity, effective survival time and T-AOC activity. Meanwhile, the added 200 U/mL SOD group showed lower MDA content and H O content. In conclusion, addition of SOD to Modena improved the boar sperm quality by reducing oxidative stress during liquid preservation at 17°C and the optimum concentration was 200 U/mL.

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis. In a previous study, we isolated EV from seminal plasma of vasectomized men, thereby excluding contributions from the testis and epididymis, and identified two distinct EV populations with diameters of 50 and 100 nm, respectively. In the current study, we comprehensively analyzed the protein composition of these two EV populations using quantitative Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In total 1558 proteins were identified. Of these, ≈45% was found only in the isolated 100 nm EV, 1% only in the isolated 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology (GO) enrichment analysis suggest that both originate from the prostate, but with distinct biogenesis pathways. Finally, nine proteins, including KLK3, KLK2, MSMB, NEFH, PSCA, PABPC1, TGM4, ALOX15B, and ANO7, with known prostate specific expression and alternate expression levels in prostate cancer tissue were identified. These data have potential for the discovery of EV associated prostate cancer biomarkers in blood.