Abstract
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.
Highlights
The origin of oocytes in ovaries of adult mammalian females has been a matter of dispute for over one hundred years
Oogenesis in vitro from ovarian surface epithelium (OSE) cells We report data from pure OSE cultures of two cases investigated either under phenol red (PhR)- or PhR+ medium conditions, and pure OSE and mixed cultures (OSE + stromal compartments; one patient) studied under both conditions
Experiment 1 (Fig. 1) Primary culture of cells scrapped from the surface of ovaries of the 47 year old female was maintained for 6 days in the Dulbecco's Modified Eagle Medium/Ham's F12 phenol red free (DMEM/F12), which was supplemented with 20% fetal bovine serum (FBS) and antibiotics
Summary
The origin of oocytes (and primary follicles) in ovaries of adult mammalian females has been a matter of dispute for over one hundred years. From a phylogenetic viewpoint, it seems contradictory that mammalian females, including humans, would evolve a uniquely retrogressive reproductive mechanism, requiring preservation of their gametes from the fetal period for up to several decades. Such long lasting preservation could cause an accumulation of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles. We have shown that mesenchymal cells in ovarian tunica albuginea (TA) differentiate into surface epithelium, a source of germ cells entering blood vessels and contributing to follicular (page number not for citation purposes)
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