Browsing by Author "Dissanayake, Keerthie"

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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.
Preimplantation embryo-derived extracellular vesicles: potential as an embryo quality marker and their role during the embryo-maternal communication
(2022-01-31) Dissanayake, Keerthie; Fazeli, Alireza, juhendaja; Jaakma, Ülle, juhendaja; Salumets, Andres, juhendaja; Kõks, Sulev, juhendaja; Tartu Ülikool. Meditsiiniteaduste valdkond
Viljatus on globaalne rahvatervise probleem, mis mõjutab miljoneid inimesi. Abistav reproduktiivtehnoloogia, sealhulgas in vitro viljastamine, on aidanud mitmeid viljatuid inimesi. Küll on sellel metoodikal üheks kitsaskohaks implantatsiooni ebaõnnestumine isegi morfoloogiliselt parimate embrüotega. Seetõttu toimuvad jätkuvalt uuringud tuvastamaks paremaid meetodeid, mis hindavad embrüo kvaliteeti ja ennustavad siirdamise edukust, olles peamiselt embrüokasvusöötme baasil. Rakuvälised ehk ekstratsellulaarsed vesiikulid (EV) on membraaniga ümbritsetud nanoosakesed, mida toodavad peaaegu kõik rakutüübid erinevates füsioloogilistes ja patoloogilistes konditsioonides. Nende kaudu toimub rakuvaheline suhtlus. Mitmed uuringud, eriti vähi korral, on uurinud EVde potentsiaali biomarkerina ja ravimkandursüsteemina. Antud doktoritöö uuris implantatsiooni-eelse perioodi embrüost vabanenud EVde potentsiaali embrüokvaliteedi markerina ja embrüo-emaka suhtluse vahendajana. Katsed viidi läbi kasutades veise-embrüoid ja inimrakukultuuride põhiseid eksperimentaalmudeleid. Esimene uuring tõestas, et individuaalselt kasvatatud implantatsiooni-eelse perioodi veise-embrüod eritavad EVsid kasvusöötmesse ning nende kontsentratsiooni- ja suurusprofiil sõltub embrüo kvaliteedist ja arengustaadiumist. Järgnevalt katsetati munajuharakkudel implantatsiooni-eelse perioodi embrüost pärit EVde funktsionaalsust. Katse käigus selgus, et EVd kõrge kvaliteediga embrüotest muutsid munajuharakkude geeniekspressiooni, mida aga ei teinud halva kvaliteediga embrüote EVd. Suurenenud ekspressiooniga geenide hulgas olid mitmed interferoon-τ raja interferooni stimuleerivad geenid. Interferoon-τ peetakse mäletsejaliste tiinuse tuvastusmolekuliks. See leid viitab, et munajuha tunneb ära kvaliteetse embrüo. Viimaseks uuriti embrüo EVde funktsionaalsuse spetsiifilisust. Leiti, et endomeetrium reageerib vaid embrüo päritolu EVdele. Uuringute käigus tuvastati embrüost vabanenud EVde potentsiaal ja spetsiifilisus embrüokvaliteedi biomarkerina.
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.