Characterization of extracellular vesicles labelled with a lipophilic dye using fluorescence nanoparticle tracking analysis

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask™ Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA

Spermatozoa, acts as an external cue and alters the cargo and production of the extracellular vesicles derived from oviductal epithelial cells in vitro

The oviduct provides optimum physiological and biochemical milieu essential for successful fertilization, early embryo development and facilitates functional maturation of spermatozoa. A study has revealed that spermatozoa alters the gene expression in bovine oviductal epithelial cells (BOECs) remotely via bio-active particles, thus acting as a cue to the oviduct prior to their arrival. However, very little attention has been paid to the question of whether spermatozoa could alter the cargo of extracellular vesicles (EVs) derived from BOECs. Therefore, the aim of this study was to investigate the alterations in small non-coding RNAs in EVs cargo derived from BOECs when incubated with spermatozoa in contact and non-contact co-culture models. After 4 h of incubation the EVs were isolated from the conditioned media, followed by small non-coding sequencing of the BOEC derived EVs. Our results revealed that EVs from both co-culture models contained distinct cargo in form of miRNA, fragmented mRNA versus control. The pathway enrichment analysis revealed that EV miRNA from direct co-culture were involved in the biological processes associated with phagocytosis, macroautophagy, placenta development, cellular responses to TNF and FGF. The mRNA fragments also varied within the different groups and mapped to the exonic regions of the transcriptome providing vital insights regarding the changes in cellular transcriptome on the arrival of spermatozoa. The findings of this study suggest that spermatozoa, in contact as well as remotely, alter the EV cargo of female reproductive tract epithelial cells which might be playing an essential role in pre and post-fertilization events

Bovine follicular fluid derived extracellular vesicles modulate the viability, capacitation and acrosome reaction of bull spermatozoa

While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice

Spermatozoa induce transcriptomic alterations in bovine oviductal epithelial cells prior to initial contact

The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 μm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival