The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice

In the current study, a decellularized ovary scaffold was used for the encapsulation and development of preantral follicles. Maturation and the expression of the related genes were evaluated and compared against in vivo conditions. Mature rat ovaries were decellularized and decellularization was confirmed using hematoxylin/eosin and Hoechst stains. Collagen and glycosaminoglycan (GAG) preservation and the extracellular matrix (ECM) architecture were evaluated by Masson’s trichrome staining, Alcian blue staining, and scanning electron microscopy, respectively. Raman confocal microscopy and DNA quantification were performed to show DNA and cell debris depletion and ECM retention. Preantral follicles of mice were cultured for 12 days in the decellularized (3D) and two-dimensional (2D) conditions. Finally, the survival rate, antrum formation rate and diameters of follicles, levels of estradiol (E2) and progesterone (P4), and expression of the ZP2, Gdf9, Bmp6, and Bmp15 genes were evaluated in the 2D and 3D culture systems. On day 13 of culture, oocyte maturation was induced by hCG. Although histochemical tests showed partial retention of ECM, Raman confocal microscopy showed some extent of ECM washing out along with the depletion of DNA and cell debris. However, SEM showed the preservation of the ovarian ECM’s ultrastructure. The survival rate of follicles was similar, but the antrum formation rate, concentrations of E2 and P4, and oocyte maturation rate were significantly higher in 3D compared with 2D. The level of ZP2, Gdf9, Bmp6, and Bmp15 gene expression in follicles matured in 2D, 3D, and in vivo was statistically similar. In all matured follicles, their gene expression levels significantly declined relative to preantral follicles. Ovarian follicles that matured in the decellularized ovaries demonstrated the same follicular growth and maturation gene expression levels as those that grew in the natural ovary. This is consistent with using the decellularized ovary as a scaffold suitable for ovarian reconstruction.Abbreviations GAG: glycosaminoglycan; ECM: extracellular matrix; 2D: two-dimensional; E2: estradiol; P4: progesterone; BMP15: bone morphogenetic protein 15; GDF9: growth differentiation factor 9; ZP2: zona pellucida 2; Gdf9: growth/differentiation factor-9; Bmp6: bone morphogenetic protein 6; Bmp15: bone morphogenetic protein 15.