ABSTRACT
INTRODUCTION Oocytes play a major role in launching the next generation either by fertilization or by nuclear transfer. Nuclear transfer is a primary requisite for embryonic stem (ES) cell production for regenerative medicine. Only a limited number of oocytes obtained from in vitro fertilization (IVF) clinics can be utilized for this purpose, hence restricting the practice of such clinical applications. In 2003, Hubner et al. were the first to report the derivation of oocytes from mouse male and female ES cells, raising anticipations of prospective unlimited source of oocyte pool. The ES cell line used in that study had a restricted germ cell expression of Oct-4 coupled with green fluorescent protein (GFP). In that way, germ cell differentiation could be followed step by step in vitro. Cells within the ES cell cultures that were maintained in leukemia inhibitory factor (LIF)-free medium with no supporting feeder cells expressed Oct-4 after four days of culture indicating on the appearance of germ cells. The proportion of cells expressing Oct-4 increased with time, and by day 8 about 40% of cells were Oct-4 positive. Large colonies with reduced Oct-4 expression but with high Vasa expression appeared at about day 12 of culture. The cells within these colonies were of three types; Oct-4-only positive cells, Oct-4-and Vasa-positive cells, and Vasa-only positive cells. This suggested that the in vitroderived germ cells were at different stages of differentiation. Culture of detached aggregates from these colonies for further two weeks resulted in the appearance of follicle-like structures from which 20% contained oocytes larger than 40 mm. The cultures were positive to estradiol and growth differentiation factor 9 (GDF-9), proposing that the follicular structures were functional and that an active interaction between the oocytes and its surrounding cells is active. By day 26, oocytes of different sizes (50-130 mm) surrounded by a fragile zona pellucida were detached from the aggregates. While the oocytes expressed the markers Figa, ZP2, and ZP3 they did not express ZP1, which elucidates the fragility of the zona pellucida. In addition, oocytes with a diameter larger than 25 mm expressed the meiotic protein SCP3, indicative of their entry into meiosis. Larger oocytes extruded the first polar body and underwent spontaneous cleavage forming parthenogenetic embryos. Following this breakthrough study, mouse and human oocyte-like cells derived from ES cells were reported by few other research groups with varying results (1-4).
