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Cassandra Matsushige C Matsushige, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, United States

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Kaelyn Kitazumi K Kitazumi, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, United States

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Amanda Beaman A Beaman, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, United States

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Marissa Miyagi M Miyagi, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, United States

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Michelle D. Tallquist M Tallquist, Department of Medicine, University of Hawaii at Manoa, Honolulu, United States

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Yukiko Yamazaki Y Yamazaki, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, United States

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We recently developed a three-dimensional (3-D) ovarian tissue culture system supported by bacterial-derived dextran hydrogel. Arg-Gly-Asp (RGD) is an extracellular matrix (ECM)-derived triple peptide. Immature ovarian tissues cultured in RGD-modified dextran hydrogel significantly promoted antral follicle growth and oocyte quality compared with those cultured in dextran hydrogel alone. In this study, we examined the mechanism of follicle growth stimulated by RGD treatment in the 3-D system. First, we detected that direct contact between RGD-modified dextran hydrogel and ovarian interstitial cells is necessary to promote antral follicle growth. Therefore, we hypothesized that RGD stimulates antral follicle growth through RGD-binding integrin receptors expressed in the interstitial cell mass. Using qPCR and immunochemical staining, we identified that integrins ⍺vβ3 and ⍺v5 are predominantly expressed in the ovarian interstitial compartment. To assess the effect of RGD-integrin interaction on follicle growth, ovarian tissues were cultured with Cilengitide (Ci), an inhibitor specific for ⍺vβ3 and ⍺vβ5. Ci treatment suppressed RGD-induced follicle growth and oocyte quality in a dose-dependent manner. When the interstitial cell aggregates were cultured with RGD, cell migration and theca-related gene expression were significantly upregulated. Ci treatment dramatically suppressed these RGD-induced activities. In co-culturing interstitial aggregate and secondary follicles with RGD, migrating cells formed outermost cell layers around the follicles, like theca layers, which were totally blocked by Ci treatment. In conclusion, our results suggest that RGD stimulates theca cell differentiation in the ovarian interstitial cells through integrins ⍺vβ3 and ⍺v5 to promote antral follicle growth in our 3-D system.

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