Formation of Ovary-Like Multitissue Spheroids Composed of Isolated Rat Follicles In Vitro

Abstract

Intact preantral follicles were isolated from immature rat ovaries by enzymatic dissociation and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. The globular shape of the follicles was maintained during culture for 96 h on agar-coated plates. When the follicles were placed near the center of the plates, follicles assembled, being in close contact with each other 24 h after culture and after 48 h large spheroidal tissues had formed. Histological examination revealed that these tissues possessed many intact follicles with preserved oocytes and many differentiated cells filled the gaps between follicles. The layer resembled squamous epithelium covered this spheroidal tissue. Culture in serum-free DMEM resulted in follicle assemblage but not formation of ovary-like multitissue spheroids (ovarioids). On the other hand, fetal calf serum induced ovarioid formation in a concentration-dependent manner as did the fraction containing the 30-100-kDa protein from fetal calf serum. Protein and RNA synthesis inhibitors inhibited ovarioid formation. The formation of ovarioids from isolated ovarian follicles may prove to be a useful model to study the ovarian cell-cell or tissue-tissue attachment and the cell differentiation in vitro.