Oogenesis in Cultures Derived from Adult Human Ovaries
May 5, 2005 - 8:38:38 PM

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.

Altogether, our observations show for the first time that granulosa cells (Figs 1e and f) and oocytes (Figs 2 and 3) may develop directly from cultured OSE cells derived from adult human ovaries. This confirms our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. In addition, the oocytes developed in vitro undergo the first meiotic division, (Fig. 5b), after which they become suitable for fertilization. Oocytes may also develop in cultures containing ovarian stromal components. We speculate that such oocytes may originate from migrating germ cells or OSE invaginations (cortical crypts, Fig. 5a). The development and maturation of oocytes appear to be stimulated by estrogens. Depending on culture conditions (type of media utilized), processing of the collected cells, age of the ovaries, commitment of neighboring cells, and other local and hormonal factors, the progenitor mesenchymal cells in ovarian cultures may differentiate into additional cell types, including granulosa cells, or persist unchanged. The ability to produce mature mammalian eggs from adult ovaries in vitro has several potential applications in the human and animal reproduction. Firstly, compared to the collection of follicular oocytes, the technique is easier (scrapping of the ovarian surface, with or without cortical component) and the yield might be higher for IVF and veterinary medicine purposes, since differentiation of primary oocytes in vitro may provide a larger number of secondary (mature) eggs. Secondly, for IVF purposes, this technique may be successful in women with premature ovarian failure, who lack follicles in their ovaries. Thirdly, the development and differentiation of oocytes from OSE precursors in vitro may help to better understand the process of oocyte renewal in vivo, and the role of accompanying granulosa and mesenchymal cells in the regulation of oocyte maturation or preservation. Fourthly, frozen OSE cells (oocyte stem cells) from younger females may be preserved for later production of fresh eggs. This may prevent the occurrence of fetal genetic alterations, which are often associated with pregnancies in advanced maternal age, possibly due to the lack of follicular renewal in aging ovaries. In addition, a colonization of premenopausal ovaries with younger oocyte and granulosa stem cells may establish a new cohort of primary follicles. This may result in a 10- to 12-year delay of the onset of natural menopause. Finally, the ovarian stem cells may serve as progenitor cells for several cell types for stem cell research, and fertilization of evolved mature human oocytes could result in the production of totipotent embryonic stem cells for research purposes and therapeutic applications.

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