Postdoctoral Research Fellow University of Toronto/CReATe Fertility Centre Toronto, Ontario, Canada
Abstract Authors: Cyntia Duval1,2; Kayla Blanchet-Plante1; Brandon A. Wyse1; Clifford L. Librach1,2,3,4,5
1 CReATe Fertility Centre, Toronto, Canada;
2 Department of Physiology, University of Toronto, Toronto, Canada;
3 Institute of Medical Sciences, University of Toronto, Toronto, Canada;
4 Department of Obstetrics and Gynecology, University of Toronto, Toronto, Canada;
5 Sunnybrook Research Institute, Toronto, Canada.
Abstract Text: Extracellular vesicles (EVs) are very important for cell-cell communication in ovarian follicles and exogenous molecules could perturb these exchanges affecting crucial events involved in oocyte maturation and competence. Indeed, cannabidiol (CBD), sometimes found in cannabis products, has been reported to decrease cancer cell exosome release. We have recently shown that cannabis metabolites (including tetrahydrocannabinol (THC) can be found in the follicular fluid of patients undergoing IVF treatment. Thus, the objectives of this study were to determine if CBD and/or THC affect EVs release by granulosa cells.
Granulosa cells were obtained from consenting patients undergoing in vitro fertilization treatments with ethics approval (IRB #16518). 0.5x106cells/mL were plated in a 6-well plate for 24h and then treated with EV inhibitors (Imidazole (10µM), GW4869 (5µM)), cannabis compounds (CBD 1µM), or a combination of THC metabolites (Δ9-THC 25ng/mL, 11-OH-THC 5ng/mL and 11-COOH-THC 50ng/mL). The next day, exosome-like particles were enriched using Total Exosome Isolation Reagent from conditioned cell culture media (Invitrogen), quantified using NanoSight NS300 and DynaPro Plate Reader III. Protein concentrations were assessed using Qubit, and exosome-like particle membrane protein content was assessed using the MACSPlex Exosome Kit (Miltenyi).
No difference in the number of cells or cell viability was observed after 24h, but a significant decrease in the number of particles and particle size 25-150nm when cells were treated with Imidazole (2.6x107/mL, p< 0.01), GW4869 (1.7x107/mL, p< 0.001) and CBD (4.2x107/mL, p< 0.01), compared to control (11.3x107/mL). In contrast, we observed a significant increase in number of particles when cells were treated with THC (18.5x107/mL, p< 0.01), compared to control (11.3x10^6/mL). When exosome-like particles were enriched, there was no difference in total protein content or exosome marker expression (CD9, CD63, and CD81). We observed a significant decrease in the expression of CD9, CD44, CD142, CD24 and CD41b in EVs enriched from GW4869 treatment (p< 0.05), but not for the other treatments (n=4).
In conclusion, CBD decreased the amount of EVs secreted while THC increased it, but there was no change in their membrane protein content. Investigations into the effect of acute and chronic exposure on EV secretion, as well as differences in protein and miRNA cargo are ongoing.
