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Background  The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated; one reason is that there is no follicle culture system that can reproduce theca cell layer formation in vitro. Therefore, a three-dimensional follicle culture system that can reproduce theca cell layer formation is required.    Methods  A collagen gel was used in the follicle culture system. To determine the optimum conditions for follicle culture that can reproduce theca cell layer formation, the effects of hormonal treatment and cell types co-cultured with follicles were examined. In addition, immunohistochemistry was used to examine the properties of the cell layers formed in the outermost part of follicles.    Results  Follicles maintained a three-dimensional shape and grew in collagen gel. By adding follicle-stimulating hormone (FSH) and co-culturing with interstitial cells, the follicles grew well, and cell layers were formed in the outermost part of follicles. Immunohistochemistry confirmed that the cells forming the outermost layers of the follicles were theca cells.    Conclusion  In this study, follicle culture system that can reproduce theca cell layer formation  in vitro  was established. In our opinion, this system is suitable for the analysis of theca cell layer formation and contributes to our understanding of the mechanisms of folliculogenesis.

Co-culturing of follicles with interstitial cells in collagen gel reproduce follicular development accompanied with theca cell layer formation