Figure 4

Morphological and molecular characterization of EVs: The size of the EVs isolated from the spent culture media of granulosa cells using NTA analysis and the mode size was between the range of 120–130 nm (A) and no significant difference was observed in the size of the EVs released from granulosa cells cultured under both temperatures (B). Immunoblotting analysis of EVs marker protein CD63, FLOT1, and TSG101 and cellular contamination indicator CYCS. The CD63, FLOT1, and TSG101 were detected in the EVs and the cellular protein lysates, However, the CYCS was not detected in the EVs, but detected only in the cellular protein sample lane. Full-length blots are presented in Supplementary Figure S6 (C). The concentration of EVs released from granulosa cells was quantified using the NTA analysis. Granulosa cells subjected to HS release significantly more EVs than the cells kept at 37 °C (D). Transmission electron microscope (TEM) shows a clear morphology of EVs (indicated by red arrows) from both the control and HS group. The upper panels indicate EVs with a scale bar of 100 nm and the lower bars indicate a scale bad of 50 nm (E). The RNA size distribution of EVs demonstrated the presence of the peak of small RNA molecules and the absence of the 18 s and 28 s cellular RNA components (F). Data are presented as mean ± SEM and the mean differences were analyzed using the Two-tail student’s t test. **p < 0.01, ns not significant.