Oogenesis in cultures derived from adult human ovaries

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.

Figure 1

Cell phenotypes in 6-day OSE culture maintained in phenol red free DMEM/F12 medium. a) Live fibroblast type cells in phase contrast microscopy (PhC) show perinuclear accumulation of organelles (arrowhead). b), Immunostaining of the same cells with HSPZ antibody shows ZP+ nuclei and perinuclear organelles (arrowhead). c) A cluster of cells showing epithelial phenotype with ZP+ nuclei and perinuclear organelles (arrowhead). d) ZP+ neural type cell shows extensions toward the ZP+ perinuclear staining (arrowhead) of neighboring mesenchymal type cells. Inset shows similar type of cell in the live culture. e) Clusters (arrows) of small (15 μm) cells of granulosa cell phenotype with strong ZP expression. Arrowhead indicates mesenchymal type cells. f) Double staining for ZP/CK5,6,8,17 (blue substrate) shows dark-blue staining of small cell clusters (arrows), but mesenchymal type cells remain brown only (arrowhead).

Figure 2

Five-day OSE culture maintained in DMEM-HG medium with phenol red. a) Phase contrast microscopy of a live cell with a large oocyte phenotype (120 μm in diameter) shows a centrally located nucleus with nucleolus and perinuclear accumulation of cell organelles. b) Larger cell reaching 180 μm shows similar morphology. c) The cell presented in panel b stained for CK18 shows cytoplasmic staining with an accumulation of CK18 immunoexpression in the perinuclear space (arrowhead). d) The same cell in CK18 (brown) and ZP (blue) double color immunostaining (CK/ZP) shows high density of ZP expression in the perinuclear space (compare with panel c). e) Control staining shows no reactions in OSE cells when the anti-HLA DR antibody was used in place of the primary antibody. f) A cluster of cells exhibiting epithelial phenotype shows nuclear ZP expression (arrowhead) and differentiation into large cells (arrows). g) Another cluster subjected to the ZP/CK,5,6,8,17 double staining shows dark-blue staining of smaller epithelial cells (arrowhead), which diminishes in more differentiated larger cells (arrow). Bar in (a) for a-e.

Figure 3

Five-day OSE culture maintained in the DMEM-HG medium with phenol red as in Fig. 2. a) Cell of oocyte phenotype with two nuclei. The centrally located nucleus shows ZP immunostaining (arrowhead) but adjacent nucleus is unstained (arrow). Note ZP+ intermediate filaments. b) The same cell subjected to double color (ZP/CK5,6,8,17) immunohistochemistry shows no additional (dark blue) staining. c) Another large cell stained for vimentin shows expression in intermediate filaments and two unstained large nuclei (arrows). d) Double color CK18/ZP staining shows intermediate filaments and ZP+ centrally located nucleus (arrowhead) and unstained adjacent structure resembling the polar body (arrow). e) Cell with centrally located ZP+ nucleus (arrowhead) and unstained fragmented adjacent structure (arrow). Note a lack of ZP+ intermediate filaments (compare with panel a) and surface ZP expression (solid arrow; compare with black arrow, panel d). f) Nuclear expression (arrowhead) of meiotically expressed PS1 carbohydrate ZP antigen. Note focally enhanced (coarse) staining (arrow) in one of the two rounded structures. g) Two cells showing a lack of nuclear PS1 expression (arrowheads), but strong staining is associated with adjacent polar-like bodies (arrows).

Figure 4

Development of oocytes in day 3 to 5 mixed ovarian cultures. Undifferentiated (stem) cells persist in all day 3 cultures (a). Under PhR+ conditions (b-h), the 100 μm oocyte-like cells were found on day 5 (arrows, b) with developing zona pellucida layer (arrow, c) in the presence of associated fibroblasts (arrowheads). Association of granulosa type cells (arrowheads, d) appears to stimulate advanced oocyte growth. Thick zona pellucida layer is apparent in some oocytes accompanied by fibroblasts on day 4 (e), and staining for ZP proteins on day 5 shows no change in the oocyte size but a lack of surface ZP expression (arrow, f). Staining for CK5,6,8,17 (g) shows cytoplasmic expression in mesenchymal type cells (black arrowhead) and non-specific staining (peroxidase expression) in the nuclei of other cells (arrowheads). h) Isolated large (200 μm) cells showed characteristics of secondary oocytes, including poor separation between the nucleus and cytoplasm (black arrowhead) expulsion of polar body (white arrowhead) and surface expression of ZP proteins (black arrow). The latter was, however, not apparent in the surface segment close to the polar body (white arrow). Under PhR- conditions, rare giant oocytes (300 μm) were found on day 4 (i). They lacked the germinal vesicle breakdown and surface expression of ZP proteins on day 5 (arrow, j). Bar in (i) for a-f and h-j.

Figure 5

Comparison of human oogenesis in vivo and in vitro a) A working model of possible pathways for oogenesis and formation of primary follicles in adult human ovaries in vivo (adjusted from Ref. [6]). (1) Ovarian tunica albuginea (ta) stem cells (green color) first differentiate into the CK+ fibroblasts (blue color) and by mesenchymal-epithelial transition give rise to the SE cells directly covering the ovarian cortex (arched arrow). (2) Closing of TA results in the formation of epithelial cords in the upper ovarian cortex. (3) Fragmented epithelial cords give rise to the epithelial nests, which resemble primitive granulosa cells and descend into the lower ovarian cortex. (4) Depending on certain in situ (stromal and neuronal) and systemic (hormonal) influences, the TA progenitors differentiate into the SE cells covering TA, which may, by asymmetric division, give rise to the ZP+ germ cells. (5) These primitive germ cells symmetrically divide, descend into the ovarian cortex, and associate with adjacent cortical vessels (6). Intravascular transport (7) is associated with a substantial increase of germ cell size and with development of ZP+ anchors (green lines), which may serve to slow down the transport speed and signal the epithelial nests to associate with a particular vascular segment. 8) The intravascular germ cells differentiating into the oocytes are picked up by epithelial nests associated with the proper cortical vessels. Such oocyte-nest complexes show an "octopus-like" (9) formations during the early stage of assembly, and a formation of the Balbiani body during the intermediate stage (light blue body, 10). The Balbiani body persists in resting primary follicles (11), but diminishes upon the growth promoting signals, including Thy-1 dp signaling derived from the follicle-accompanying vessels (12, dashed line). An alternative pathway for the germ cell origin from TA precursors (4') consists of a constitution of cortical crypts formed by SE-like embryonal type cells, possibly originating from, but not necessarily connected with the deep SE invaginations, as evidenced from serial sections. The "alternative" pathway of germ cell origin may supply the oocytes directly to the neighboring nests (dashed arched arrows) and, via vascular transport (dotted arched arrow), saturate distant nests to form the primary follicles. The oocytes not utilized for follicular renewal accumulate in medullary vessels and degenerate (bottom right) [6]. b) In vitro, the CK5+ oocyte precursors may differentiate into CK5- cells (see Fig 2g), enlarge and undergo germinal vesicle breakdown followed by first meiotic division with a release of the polar body (pb) and ZP surface expression (brown color, see Fig. 3e and 4h).