Studies on the development of culture system for primary follicle-derived embryonic stem cells in mice

Abstract

This study examined the establishment of culture system for in-vitro culture of primary follicles and the difference of some cytosolic organelles in mature oocytes retrieved from different origins in mice. First, appropriate retrieval time of primary follicles was evaluated. The primary follicles retrieved from neonatal females at different ages (7 to 14-day-old) were cultured for obtaining mature oocytes, and assessment on the efficiency of follicle retrieval and mature oocyte generation was subsequently made by employing both cellular and biological parameters. The number of primary follicles was significantly increased from 9 up to 11-day-old mice. When cultured in vitro, primary follicles retrieved from 11-day-old mice showed best follicular development and the largest number of mature oocytes per euthanized animals. In conclusion, primary follicles could be cultured in vitro and 11-day-old mice were appropriate as primary follicle donors.

FSH is essential for follicular development such as viable oocyte recovery, oocyte maturation, granulosa cell proliferation, formation of antral-like cavities and cumulus cell expansion. To optimize culture condition for primary follicles, I attempted to decide proper combination of FSH concentration. Morphology of primary follicles, follicular development, oocyte maturation, steroid hormone production and receptor expression of granulosa cells were determined. The best follicular development and blastocysts formation after IVF and parthenogenetic activation were observed in 200 mIU/ml FSH-treated group during the whole culture period. When FSH was added to culture media, normal follicular development, steroid hormone production, expression of hormone receptors were observed. In conclusion, I established optimal primary follicle-culture system for producing mature oocytes and viable homozygotic, heterozygotic blastocysts.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) of blastocyst through in vitro culture on MMC-treated feeder layer under appropriate culture conditions. Because of the developmental potential to differentiate into any type of cells which consist of human body, ESCs have been getting spotlight as the clinical purpose such as stem cell therapy. Next, I established several ESC lines from homozygotic and heterozygotic blastocysts derived from primary follicles cultured under optimal condition. To confirm several characters of colony-forming cells as embryonic stem cells, I conducted conventional characterization methods such as ESCs-specific marker staining, expression of stemness-related genes and in vitro and in vivo differentiation. In conclusion, established cell lines have suitable chracteristics as stem cells and can be utilized to reproductive medicine and stem cell biotechnology.

Finally, I demonstrated that retrieval age and in vitro -culture condition may affect the quality of mature oocytes and afterward developmental competency. In case of mature oocytes derived from early secondary follicles of 8-week-old mice could not develop to the blastocyst stage after parthenogenetic activation. By the analysis of ultrastructure through TEM, the differences in cytosolic organelles among mature oocytes from different origins were observed. These differences may affect the development of embryo and make the distinction among groups. In conclusion, combined stabilized culture protocol of primary follicles and cytoplasmic study will contribute to develop novel artificial reproductive technology and establish experimental model system for elucidating the mechanism of follicular development.