Comparison of follicle formation and molecular characteristics in reconstituted ovarioids under limited somatic cell conditions

Reconstructed ovaries are emerging as a promising approach for assisted reproduction and species preservation due to their potential for in vitro follicle formation and oocyte production. However, efficient production of reconstructed ovaries remains technically limited by the requirement for securing a large number of gonadal somatic cells. Therefore, this study evaluated the feasibility of follicle formation under limited somatic cell conditions by forming ovarioids with a reduced number of somatic cells. The formation of follicle-like structures was confirmed using immunofluorescence analysis, and the expression of molecular markers related to apoptosis, autophagy, mitochondrial stability, and stress responses was analyzed. As a result, it was observed that the groups with a reduced number of gonad somatic cells (2000:20000 and 3000:30000) formed follicle-like structures similar to the control group. Furthermore, oocytes observed under all conditions ranged in diameter from 50 to 70 µm, corresponding to the primary oocyte stage. Additionally, it was observed that the microenvironment was partially reproduced through staining of LAMININ, N-CADHERIN, and CD44. However, as the number of somatic cells decreased, differences were observed in the expression patterns of follicle-related and stress-related molecules. In particular, compared to the control group, the 2000:20000 group showed a significant decrease in follicle-related factors (Nobox, Lhx8, Gdf9, Bmp15). These results suggest that the number of somatic cells and the composition of the microenvironment in a reconstructed ovarian model can affect the qualitative environment for follicle formation, providing a basic basis for optimizing the conditions for ovarian reconstitution.