A dynamic culture system to support the in vitro growth and maturation of ovarian follicles.

摘要

Ovarian follicle growth is a prolonged process that involves progressive development of the follicle unit through specific histologically defined stages of development. Groups around the world have begun ovarian cryopreservation programs for young girls and women undergoing potentially sterilizing surgery or chemotherapy with the hope that follicles can be isolated from these tissues and grown in vitro at a later date. Though follicles derived from mice can be grown up to maturity using conventional culture techniques, scientists have been unsuccessful with the in vitro development of follicles from species that have larger follicles. The objective of this study was to develop a culture system that could better support the growth and maturation of these larger follicles. The aims of the study focused on maintaining structural integrity through a suspension culture technique, providing three-dimensional support by utilizing an alginate microencapsulation technique, and creating a unique oxygen environment that more closely mimicked the oxygen levels of the native ovary. The suspension culture technique was found to eliminate follicle flattening that occurred with larger follicles on flat surfaces in a static culture. The alginate microencapsulation technique was shown to improve the support of three-dimensional growth of preantral follicles; but requires the inclusion of FSH in the scaffold in order to maintain the growth rate of unencapsulated follicles. Finally, by implementing a dynamic oxygen protocol based on the unique oxygen environment of the ovary, both the yield and quality of the oocytes derived from in vitro cultured preantral follicles were significantly improved when compared to oocytes from follicles cultured at the traditional ambient oxygen levels. In addition, these oocytes were not only able to undergo parthenogenetic activation, but were also fertilized through intracytoplasmic sperm injection. A subsequent gene expression analysis uncovered that follicles grown in a high oxygen environment possessed more differentially expressed genes compared to an in vivo control than did follicles cultured in a low oxygen environment. Furthermore, these differentially expressed genes were found to regulate several key processes that contribute to proper follicular development. These findings have contributed to the development of a novel culture system that has enhanced the in vitro support of follicle and oocyte maturation.