Decellularized ovarian bioscaffolds and resveratrol-loaded polymeric nanoparticles improve in vitro development of bovine preantral follicles

This study evaluated the effects of decellularized ovarian bioscaffolds and resveratrol-loaded polymeric nanoparticles (RLPN) on the in vitro development of bovine secondary follicles. Decellularized extracellular matrix (dECM) from cortical fragments was obtained by freeze–thaw cycles and sequential incubation in Triton X-100 and sodium dodecyl sulfate. Decellularization efficiency and extracellular matrix integrity were assessed by hematoxylin-eosin, Hoechst staining, scanning electron microscopy, and quantification of collagen and glycosaminoglycans. Bovine secondary follicles were isolated and cultured for 12 days in either a two-dimensional (2D) system or in dECM scaffolds in medium supplemented with 0.02, 0.2, or 2.0µM RLNP, blank nanoparticles, or unencapsulated resveratrol. Follicular viability and ultrastructure were evaluated by calcein-AM/ethidium homodimer-1 staining and transmission electron microscopy. Expression of mRNA for catalase, superoxide dismutase, glutathione peroxidase 1, peroxiredoxin 6, and nuclear factor erythroid 2-related factor 2 was assessed by qRT-PCR. Quantitative data were analyzed by unpaired t-tests or one-way ANOVA, followed by Tukey’s test (P < 0.05). Hematoxylin-eosin and Hoechst staining confirmed effective cell removal, while collagen, glycosaminoglycans, and ECM ultrastructure were preserved. Follicles cultured in the three-dimensional (3D) system showed increased viability, further enhanced by 0.02 or 2.00 µM RLPN. Follicles cultured with 0.02 µM RLPN exhibited well-preserved morphology, including intact zona pellucida, oocyte membrane, and organelles. RLPN downregulated the expression of antioxidant genes. In conclusion, the decellularization protocol effectively removed cellular content and preserved ECM structure and ultrastructure. 3D culture system in combination with medium supplemented with 0.02 µM RLPN supported follicular development and ultrastructure, as well as downregulated antioxidant gene expression.