The Effect of Pasteurized Milk Extracellular vesicles on Bacterial Growth : [poster]

Milk-derived extracellular vesicles (mEVs) have gained attention for their potential biological activities, including antibacterial properties and they can carry bioactive molecules. The mEVs can survive high temperatures and digestion processes while retaining their biological activity. Studies of mammalian EVs are increasingly attracting the interest of researchers; however, there are only a few studies of mEVs’ antimicrobial effect.Poster of the Conference COMBIVET & OH-BOOST 2023, Tartu, Estonia, 8-9 th of September, 2023.This research was partially supported by the Estonian Research Council grant PRG 1441.This research was partially supported by the Estonian Research Council grant PRG 1441

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

Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p Peer reviewe

Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk

Background Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.Peer reviewe

Rakuväliste vesiikulite vahendatud embrüo-emaka suhtlus – vahend hindamaks implantatsiooni-eelse embrüo talituslikku pädevust

Väitekirja elektrooniline versioon ei sisalda publikatsiooneViljatus on ülemaailmne probleem, mis vaevab hinnanguliselt 15% paaridest. Viljatuse raviks kasutatakse kunstliku viljastamist, mis aitab mitmetel paaridel rasestuda. Abistatud reprodiuktiivtehnoloogia metoodikate valik on mitmekesine, kuid kõige kasutatavam on in vitro viljastamine (IVF). Alates 1970-ndatest on abistatud reproduktsioonitehnoloogia märgatavalt arenenud ja aidanud ilmale tuua miljoneid lapsi. Sellest hoolimata on IVF-i edukus alla 40%. Edutu implantatsioon on ebaõnnestunud embrüo siirdamise peamine põhjus 2/3 juhtudest. Nidatsioon on inimese paljunemise üks olulisemaid etappe, kus embrüo kinnitub endomeetriumi. Selleks peab aga endomeetriumis toimuma mitmed muutused. Üheks endomeetriumi muutuste vallandajaks peetakse embrüo ja endomeetriumi vahelist suhtlust. Mitmete rakkudevaheliste suhtluste meetodite hulgas on rakuvälised ehk ekstratsellulaarsed vesiikulid (EV) üks olulisim transportmehhanism. EV on nanoosakesed, mis sisaldavad mitmeid bioloogiliselt aktiivseid molekule. Neid molekule viivad EV ühest rakust teise. Doktoritöös uuriti inimese ja veise embrüonaalse RNA, peamiselt mittekodeeriva RNA, ülekandumist endomeetriumi rakkudelt eralduvatest EV-dest. Embrüo RNA märgistamisega kinnitati, et embrüonaalne RNA kandub endomeetriumi rakkudesse. Selline rakkudevaheline suhtlus muutis endomeetriumi rakkude vastuvõtlikkust embrüo implantatsiooniks, millega tõestati et embrüo EV võivad märkimisväärselt muuta endomeetriumi rakkude talitlust nii inimesel kui ka veisel. Kunstlikus viljastamises on üha populaarsem siirata vaid üks parima kvaliteediga embrüo, kuid hindamismeetodid embrüo valimiseks on puudulikud. Antud doktoritöös leiti, et endomeetriumi rakkudes (eriti endomeetriumi transkriptoomis) toimunud muutused olid seoses embrüo kvaliteediga. Seda tulemust embrüo-emaka suhtluses saab teoreetiliselt kvantifitseerida ja kasutada mitteinvasiivse meetodina hindamaks embrüo kvaliteedi, mis parendaks viljatuse ravi edukust ja vähendaks patsientide kannatusi.Subfertility is a global condition with estimated 15% of couples worldwide have trouble conceiving. In many of these cases, the most effective remedy is assisted reproduction. Assisted reproduction can take many forms. In vitro fertilization or IVF being the one with which the most people are familiar. Since its conception in the 1970s, assisted reproduction has developed significantly producing millions of successful pregnancies. However, the rate of success of IVF remains below 40% even in ideal candidates. Failure of implantation has been identified as the main cause of failed embryo transfers in 2/3 of the cases. Implantation is one of the most critical steps in human reproduction. The embryo attaches itself to the endometrial epithelium. The endometrium changes drastically in anticipation of the incoming embryo. One trigger that brings about this change is thought to be the communication between embryo and the endometrium. Among many available methods of intercellular communication, extracellular vesicles stand out because of its remarkable utility as a cargo carrier. Extracellular vesicles or EV are nanometre sized sacs that contain biologically active molecules. They can transfer these molecules in between cells as cellular messengers. In this project, we have investigated the phenomenon of embryonic RNA, especially non-coding RNA, transfer to endometrial cells using EVs as a transport mechanism. By labelling embryo RNA, we were able to confirm that Embryonic RNA is in fact transferred to the endometrium. This intercellular communication changes the physiology of the endometrium in such a way that endometrial cells were transformed into a higher level of receptivity for the incoming embryo. We also observed that only embryo EVs can induce this remarkable change on the endometrial cells. The embryonic EV based communication was also observed in bovine embryo-maternal interactions. Interestingly, the changes induced in the endometrial cells, especially in the endometrial transcriptome, correlated with the quality of the embryo that produced the EVs leading us to theorize that this method of quantifying the embryo-maternal communication can be used as a non-invasive method of embryo grading. In the ever-popular practise of elective single embryo transfer in assisted reproduction, selecting the best embryo is hugely advantages. Quantified embryo maternal communication could therefore be an invaluable tool in the field of assisted reproduction to reduce patient suffering and to increase the rate of successful pregnancies.https://www.ester.ee/record=b549778

Rhythm of the First Language: Dynamics of Extracellular Vesicle-Based Embryo–Maternal Communication in the Pre-Implantation Microenvironment

One of the most critical steps in mammalian reproduction is implantation. Embryos with an impaired capacity for embryo–maternal crosstalk are thought to have a reduced potential for implantation. One agent of embryo–maternal communication is extracellular vesicles (EV). EVs are lipid bilayer-bound biological nanoparticles implicated in intercellular communication between many of the known cell types. In the current study, we isolated EVs from trophoblast analogue JAr spheroids and supplemented the EVs with receptive endometrium analogue RL95-2 cells to simulate pre-implantation embryo–maternal dialogue. The transcriptome of the endometrial cells was examined at 30 min, 4 h and 48 h intervals using Oxford Nanopore® technology. At the time points, 30 min, 4 h and 48 h, the endometrial cells showed a significantly altered transcriptome. It seems trophoblast EVs induce a swift and drastic effect on the endometrial transcriptome. The effect peaks at around 4 h of EV supplementation, indicating a generalized effect on cell physiology. Alterations are especially apparent in biological pathways critical to embryonic implantation, such as extracellular matrix–receptor interactions and cytokine–receptor interactions. These observations can be helpful in elucidating the dynamics of embryo–maternal communication in the pre-implantation period

The effect of pasteurized milk extracellular vesicles on bacterial growth

Extracellular vesicles (EVs) are released by cells and have a lipid bilayer structure. EVs harbor various molecules, including proteins, RNAs, and DNAs. Studies of mammalian EVs are increasingly attracting the interest of researchers; however, there are only few studies of nanoparticles in food. Milk-derived EVs can survive high temperatures and digestion process, while retaining their biological activity. This study investigated the effect of pasteurized cows’ milk derived EVs on growth of five different bacteria. Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 53868, Bacillus subtilis, Bacillus cereus and Pseudomonas aeruginosa with a concentration of 1x107 CFU/ml were separately co-cultured with pasteurized milk derived EVs (1x109 EVs/μl) in Muller Hinton broth. The bacterial growth was monitored as absorbance at 620 nm over 24 hours. Bacteria treated with phosphate buffer solution (PBS) were considered negative control throughout the experiment. The percentage bacterial growth difference was COMBIVET & OH-BOOST JOINT CONFERENCE 2023 55 determined with respect to negative control and results expressed as mean ± standard error of mean. All analyses were performed in three biological triplicates and each biological replicate consisted of three technical replicates. Co-culture of bacteria with milk EVs demonstrated that EVs could decrease the growth of S. aureus, B. subtilis, B. cereus and P. aeruginosa. Highest growth inhibition was observed for B. subtilis (33.9% ± 2.4) followed by B. cereus (18.1% ± 3.7) at 6 hours of incubation. S. aureus and P. aeruginosa growth were inhibited by 12.9% ± 1.3 and 9.9% ± 3.5 respectively after 9 hours incubation. Only the S. aureus growth inhibition at 9 hours was statistically significant (P=0.022) according to T-test. However, E. coli growth was not affected by milk EVs compared to the control. In conclusion, the dietary EVs can be absorbed by bacteria and pasteurized milk derived EVs has a selective inhibitory activity on the growth of some bacteria

The Extracellular Vesicles Proteome of Endometrial Cells Simulating the Receptive Menstrual Phase Differs from That of Endometrial Cells Simulating the Non-Receptive Menstrual Phase

Successful embryo implantation into a receptive endometrium requires mutual endometrial-embryo communication. Recently, the function of extracellular vehicles (EVs) in cell-to-cell interaction in embryo-maternal interactions has been investigated. We explored isolated endometrial-derived EVs, using RL95-2 cells as a model of a receptive endometrium, influenced by the menstrual cycle hormones estrogen (E2; proliferative phase), progesterone (P4; secretory phase), and estrogen plus progesterone (E2P4; the receptive phase). EV sized particles were isolated by differential centrifugation and size exclusion chromatography. Nanoparticle tracking analysis was used to examine the different concentrations and sizes of particles and EV proteomic analysis was performed using shotgun label-free mass spectrometry. Our results showed that although endometrial derived EVs were secreted in numbers independent of hormonal stimulation, EV sizes were statistically modified by it. Proteomics analysis showed that hormone treatment changes affect the endometrial EV’s proteome, with proteins enhanced within the EV E2P4 group shown to be involved in different processes, such as embryo implantation, endometrial receptivity, and embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs