Browsing by Author "Viil, Janeli"

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Bovine follicular fluid and extracellular vesicles derived from follicular fluid alter the bovine oviductal epithelial cells transcriptome."
(MPDI, 2020-07-28) Hasan, Mohammad Mehedi; Viil, Janeli; Lättekivi, Freddy; Ord, James; Ul Ain Reshi, Qurat; Jääger, Kersti; Velthut-Meikas, Agne
While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.
Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries
(MPDI, 2020-12-15) Rooda, Ilmatar; Hasan, Mohammad Mehedi; Roos, Kristine; Viil, Janeli; Andronowska, Aneta; Smolander, Olli-Pekka; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza; Velthut-Meikas, Agne
Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.
Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers
(2020-06) Dissanayake, Keerthie; Nõmm, Monika; Lättekivi, Freddy; Ressaissi, Yosra; Godakumara, Kasun; Lavrits, Arina; Midekessa, Getnet; Viil, Janeli; Bæk, Rikke; Møller Jørgensen, Malene; Bhattacharjee, Sourav; Aneta, Andronowska; Salumets, Andres; Jaakma, Ülle; Fazeli, Alireza
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 < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.
Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk
(BioMed Central Ltd, 2019-11-14) Es-Haghi, Masoumeh; Godakumara, Kasun; Häling, Annika; Lättekivi, Freddy; Lavrits, Arina; Viil, Janeli; Andronowska, Aneta; Nafee, Tamer; James, Victoria; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza
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.
Spermatozoa induce transcriptomic alterations in bovine oviductal epithelial cells prior to initial contact
(2020-09-03) Qurat Ul Ain, Qurat; Viil, Janeli; Ord, James; Lättekivi, Freddy; Godakumara, Kasun; Hasan, Mohammad Mehedi; Nõmm, Monika; Jääger, Kersti; Velthut-Meikas, Agne; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza
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.
Studies on cellular and molecular mechanisms that drive normal and regenerative processes in the liver and pathological processes in Dupuytren’s contracture
(2016-07-01) Viil, Janeli; Jaks, Viljar, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond.
Maks on imetaja suurim siseorgan, mis omab unikaalset taastumisvõimet, kuid pidev krooniline maksakahjustus põhjustab tema regeneratsioonivõime vähenemist, fibroosse armkoe tekkimist ja maksa funktsioonide kadumist. Sageli on lõppstaadiumis oleva kroonilise maksahaiguse ainsaks ravivõimaluseks maksasiirdamine. Leidmaks maksasiirdamisele alternatiivseid ravimeetodeid, on oluline uurida maksa regeneratsiooni ja haiguslike protsessidega seotud molekulaarseid ja rakulisi mehhanisme. Käesolevas töös uurisime, kuidas maksa aeglaselt jagunevad rakud panustavad koe taastamisse, millised rakuvälise maatriksi muudatused leiavad aset vastusena maksakahjustusele ja kuidas need võiksid mõjutada rakkude käitumist. Samuti uurisime, kas signaalirajad, mis on olulised maksafibroosis, omavad rolli ka teist tüüpi fibrootilises haiguses ja lõpuks viisime läbi suuremahulise kemikaalide skriinimise, leidmaks AKT1-PDPK1 interaktsiooni inhibiitorit, mis vähendaks kasvajate-ja fibroosiseoselise AKT1 aktivatsiooni. Töö tulemusena leidsime, et maksa aeglaselt jagunevad rakud on unipotentsed sapijuharakud, mis aktiveeruvad ainult kroonilise kahjustuse korral. Samuti selgus, et maksa rakuvälise maatriksi muutused sõltuvad kahjustuse tüübist ning erinevad maatriksi komponendid omavad hepatotsüütide ja sapijuharakkude proliferatsioonile erinevat mõju. Uurides fibrootilisi protsesse Dupuytren´i kontraktuuri koes, avastasime, et haige koe erinevad komponendid sünteesivad erinevaid fibroosi ja proliferatsiooni soodustavaid molekule, moodustades haiguse arenguks sobiliku keskonna. Töö viimases osas kirjeldasime AKT1-PDPK1 interaktsiooni inhibiitori, väikesemolekulaarse ühendi NSC156529, identifitseerimise protsessi. NSC156529 vähendas AKT valgu ja tema sihtmärkvalkude aktiivsust ning pidurdas rakkude kasvu in vitro ja tuumori kasvu in vivo. Seega on NSC156529 sobiv alusmolekul uudsete ravimite väljatöötamiseks selliste haiguste nagu kasvajad ning fibroos vastu, mille puhul AKT signaaliraja aktiivsus on kõrgenenud.
Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability
(ACS Publications, 2020-06-30) Midekessa, Getnet; Godakumara, Kasun; Ord, James; Viil, Janeli; Lättekivi, Freddy; Dissanayake, Keerthie; Kopanchuk, Sergei; Rinken, Ago; Aneta, Andronowska; Bhattacharjee, Sourav; Rinken, Toonika; Fazeli, Alireza
Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.