Trofoblasti rakuliini HTR-8/SVneo mikrovesiikulite ja eksosoomide sisenemine endomeetriumi rakuliinide RL95-2 ja HEC-1A rakkudesse

Nii retseptoorse kui ka mitteretseptoorse endomeetriumi kartsinoomi rakuliinide rakud võtavad sisse trofoblasti rakuliini HTR8/SVneo rakkudest pärit mikrovesiikuleid ja eksosoome, kusjuures HEC-1A rakkudesse mikrovesiikulid sisenevad põhimõtteliselt makropinotsütoosi ja klatriin-vahendatud endotsütoosi teel. RL95-2 rakkude kohta ei saa praegu öelda, mis mehhanismi mikrovesiikulid kasutavad rakkudesse sisenemiseks. Kuid kuivõrd tegemist on kasvajaliste rakkudega, siis ei ole saadud tulemuste põhjal võimalik öelda, kas see kehtib ka normaalsete rakkude puhul. Edasises töös tuleb siiski arvestada, et normaalsete trofoblastide ja endomeetriumirakkude kasvatamine on väga keeruline ning siiani puuduvad andmed kuivõrd efektiivselt toimub nendest rakkudest vesiikulite sekreteerimine

Chemosensitivity and chemoresistance in endometriosis - differences for ectopic versus eutopic cells

Research question: Endometriosis is a common gynaecological disease defined by the presence of endometrium-like tissue outside the uterus. This complex disease, often accompanied by severe pain and infertility, causes a significant medical and socioeconomic burden; hence, novel strategies are being sought for the treatment of endometriosis. Here, we set out to explore the cytotoxic effects of a panel of compounds to find toxins with different efficiency in eutopic versus ectopic cells, thus highlighting alterations in the corresponding molecular pathways. Design: The effect on cellular viability of 14 compounds was established in a cohort of paired eutopic and ectopic endometrial stromal cell samples from 11 patients. The biological targets covered by the panel included pro-survival enzymes, cytoskeleton proteins, the proteasome and the cell repair machinery. Results: Protein kinase inhibitors GSK690693, ARC-775 and sorafenib, proteasome inhibitor bortezomib, and microtubuledepolymerizing toxin monomethyl auristatin E were more effective in eutopic cells. In contrast, 10 mu mol/l of the anthracycline toxin doxorubicin caused cellular death in ectopic cells more effectively than in eutopic cells. The large-scale sequencing of mRNA isolated from doxorubicin-treated and control cells indicated different survival strategies in eutopic versus ectopic endometrium. Conclusions: Overall, the results confirm evidence of large-scale metabolic reprogramming in endometriotic cells, which underlies the observed differences in sensitivity towards toxins. The enhanced efficiency of doxorubicin interfering with redox equilibria and/or DNA repair mechanisms pinpoints key players that can be potentially used to selectively target ectopic lesions in endometriosis.Peer reviewe

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