Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.

The in vitro culture of immature ovarian follicles is used to examine the factors that regulate follicle development and may ultimately provide options for reproductive infertility. The objective of this study was to develop a three-dimensional in vitro culture system for the growth and development of individual granulosa cell-oocyte complexes. An alginate hydrogel was used to encapsulate immature mouse granulosa cell-oocyte complexes (GOCs) that were subsequently maintained in a serum-free in vitro culture. An overall incorporation efficiency of 50% was achieved. The complexes were assessed by transmission electron microscopy for changes in ultrastructure during in vitro growth. The architecture of the follicular complex was maintained during the encapsulation and the subsequent culture. The granulosa cells proliferated, and the oocytes also grew in volume and obtained the structural characteristics of mature oocytes including cortical granule formation, a well-developed zona pellucida with microvilli, normal mitochondria, and lattice-like structures in the cytoplasm. Oocytes retrieved and matured were able to resume meiosis, a necessary step for proper development. Thus, this system represents a new in vitro methodology for growth of individual granulosa cell-oocyte complexes.