Early-stage follicle development regulated by RGD peptide and ovarian fragment size in three-dimensional culture

The tripeptide Arg-Gly-Asp (RGD) is a minimal integrin-binding motif found in multiple extracellular matrix (ECM) proteins. We previously determined that immature ovarian fragments cultured in the RGD-immobilized dextran hydrogel significantly promoted antral follicle development compared to those cultured in the dextran hydrogel control. In this study, using neonatal mouse ovaries, we examined the effects of RGD peptide and ovarian fragment size on early-stage follicle development in our 3D culture system. One neonatal ovary was cut into 1/2, 1/4, or 1/8 fragments and cultured in the dextran hydrogel with or without RGD. Follicle development progressively increased as fragment size decreased (intact < 1/2 < 1/4 < 1/8). Notably, RGD treatment further enhanced follicle development in a size-dependent manner. In particular, the RGD-treated 1/8 fragments significantly promoted follicle development compared to all other groups with or without RGD. Previous studies have shown that ovarian fragmentation stimulates early-stage follicle development through disruption of the Hippo pathway. We found that the RGD treatment further increased the Hippo pathway disruption in the 1/8 fragments compared to those cultured without RGD. As a ligand, RGD binds to integrin receptors to induce various cell functions. In the neonatal ovary, ovary-specific RGD-binding integrins αvβ3 and αvβ5 were predominantly localized in the ovarian interstitial/theca cells. To evaluate the role of the RGD-integrin interaction, we blocked integrins αvβ3 and αvβ5 using Cilengitide (Ci). In the presence of Ci, RGD-induced follicle development and the Hippo pathway disruption were completely suppressed in the 1/8 fragments in 3D culture. These results suggest that the RGD-integrin interaction is crucial for promoting early-stage follicle development in 3D culture. Overall, our study demonstrates that ovarian fragment size significantly influences early-stage follicle development in culture. Fragmentation exposes the ovarian stromal compartment expressing RGD-binding integrins to the surrounding hydrogel. Moreover, the effect of RGD treatment on follicle development depends on ovarian fragment size in our 3D culture system, with 1/8 fragments yielding the greatest enhancement.