Assessing the effect of adipose tissue-derived stem cell-conditioned medium on follicles and stromal cells in bovine ovarian tissue culture

Research question Does adipose tissue-derived stem cell-conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro? Design Bovine ovaries (n=8) were sectioned and in vitro-cultured for 8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT+CM D8). Half of the culture medium was replaced every other day, and stored for estradiol (E2) production measurement. Follicle classification was established using hematoxylin and eosin (H&E) staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a follicle quality marker. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and transforming growth factor beta 1 (TGF-β1), were evaluated in ASC-CM in order to appraise potential underlying ASC mechanisms of action. Results OT+CM D8 showed significantly higher secondary follicle proportions (p=0.02) than OT Ctrl D8. OT+CM D8 also demonstrated significantly lower follicle (p=0.014) and stromal cell (p=0.001) TUNEL-positive percentages than OT Ctrl D8. Furthermore, follicles in OT+CM D8 exhibited a significant increase (p=0.002) in GDF-9 expression compared to those in OT Ctrl D8, and E2 production was significantly higher (p=0.04) after 8 days in OT+CM. All studied factors were found to be present in the ASC-CM. VEGF and IL-6 were the most widely expressed, while TGF-β1 showed the lowest concentrations. Conclusions Addition of ASC-CM to culture medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects.