Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Journal name:
Nature Medicine
Volume:
18,
Pages:
413–421
Year published:
DOI:
doi:10.1038/nm.2669
Received
Accepted
Published online

Abstract

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

At a glance

Figures

  1. FACS-based protocol for OSC isolation.
    Figure 1: FACS-based protocol for OSC isolation.

    (a) Immunofluorescence analysis of Ddx4 expression (green; with a blue DAPI counterstain) in young adult mouse ovaries using antibodies against the N (NH2) or the C (COOH) terminus of the protein (scale bars, 50 μm). (b) Immunomagnetic sorting of dispersed mouse ovaries or isolated oocytes using antibodies against the N or C terminus of Ddx4. Fraction 1, cells plus beads before separation; fraction 2, wash fraction (non-immunoreactive); fraction 3, bead fraction (Ddx4-positive cells, which are highlighted by white arrows) (scale bars, 50 μm). (c) FACS plots from the analysis of live or permeabilized cells from dispersed mouse ovaries using antibodies against the N or C terminus of Ddx4. Viable Ddx4-positive cells were only detected with the COOH antibody (red dashed box, reacted with anti-rabbit-APC), whereas permeabilization enabled the isolation of Ddx4-positive cells using the NH2 antibody (blue dashed box, reacted with anti-goat-488). (d) Permeabilization of viable Ddx4-positive cells obtained with the COOH antibody (red dashed boxes) enabled re-isolation of the same cells by FACS using the NH2 antibody (blue dashed box), as shown by the shift in fluorescence (the red to blue color indicates the shift from APC-positive live cells to 488-positive fixed and permeabilized cells). (e) Schematic of FACS protocols used to validate the Ddx4 COOH antibody for the isolation of viable OSCs. (f) Expression analysis of germline markers (Prdm1, Dppa3, Ifitm3, Tert, Ddx4 and Dazl) and oocyte markers (Nobox, Zp3 and Gdf9) in each cell fraction produced during the ovarian dispersion process to obtain cells for FACS-based isolation of OSCs. +ve, Ddx4-positive viable cell fraction after FACS; −ve, Ddx4-negative viable cell fraction after FACS; no RT, PCR of RNA sample without reverse transcription; β-actin, sample loading control. Samples were resolved through agarose gels and stained with ethidium bromide to visualize DNA bands, with the resultant images inverted for photography and display purposes.

  2. Isolation of OSCs from adult mouse and human ovaries.
    Figure 2: Isolation of OSCs from adult mouse and human ovaries.

    (a,b) Representative histological appearance of adult ovarian tissue used for the isolation of human (a) and mouse (b) OSCs. Scale bars, 100 μm. (c,d) Morphology of viable cells isolated by FACS based on cell-surface expression of DDX4 (human, left) or Ddx4 (mouse, right). Scale bars, 10 μm; insets show a twofold higher magnification of individual OSCs. (e) Gene expression profile of the starting ovarian material and freshly isolated OSCs showing an assessment of three different subjects as examples for the human ovarian tissue analyses. Samples were resolved through agarose gels and stained with ethidium bromide to visualize DNA bands, with the resultant images inverted for photography and display purposes. (f,g) Teratoma formation assay showing an absence of tumors in NOD-SCID mice 24 weeks after receiving injections of freshly isolated mouse OSCs (f) compared with development of tumors in mice 3 weeks after the injection of an equivalent number of mouse ESCs (g; with the tumor highlighted by a black-dashed oval). (h) Examples of cells from all three germ layers in a representative ESC-derived teratoma, with a neural rosette highlighted at the same magnification in the inset of the left panel (scale bars, 100 μm).

  3. Mouse OSCs generate functional eggs after intraovarian transplantation.
    Figure 3: Mouse OSCs generate functional eggs after intraovarian transplantation.

    (a,b) Examples of growing follicles containing GFP-negative (a) and GFP-positive (b) (brown; with a blue hematoxylin counterstain) oocytes in ovaries of wild-type mice injected with GFP-expressing OSCs 5–6 months before analysis. (c) Examples of ovulated GFP-negative eggs (in cumulus-oocyte complexes) and the resultant embryos (embryos at the two-cell (2-cell), four-cell (4-cell), and blastocyst (B) stages are shown as examples) generated by IVF after induced ovulation of wild-type female mice that received intraovarian transplantation of GFP-expressing OSCs 5–6 months before analysis. Scale bars, 30 μm. (d,e) Examples of GFP-positive eggs (in cumulus-oocyte complexes) obtained from the oviducts after induced ovulation of wild-type female mice that received intraovarian transplantation of GFP-expressing OSCs 5–6 months before analysis. These eggs were fertilized in vitro using wild-type sperm, resulting in two-cell embryos that progressed through preimplantation development (examples of GFP-positive embryos at the two-cell, four-cell, eight-cell (8-cell), compacted morula (CM), expanded morula (EM), blastocyst and hatching blastocyst (HB) stages are shown) to form hatching blastocysts 5–6 d after fertilization. Scale bars, 30 μm.

  4. Evaluation of mouse- and human-ovary-derived OSCs in defined cultures.
    Figure 4: Evaluation of mouse- and human-ovary–derived OSCs in defined cultures.

    (ad) Assessment of OSC proliferation by dual detection of Ddx4 or DDX4 expression (green) and BrdU incorporation (red) in mouse (a,b) and human (c,d) OSCs maintained in MEF-free cultures. Scale bars, 30 μm. (e) Typical growth curve for MEF-free cultures of mouse OSCs after passage and after seeding 2.5 × 104 cells in each well of 24-well culture plates. (f) FACS analysis using the COOH antibody to detect cell-surface expression of Ddx4 in mouse OSCs after 18 months of propagation. (g) Gene expression profile of starting ovarian material and cultured mouse and human OSCs after 4 or more months of propagation in vitro. Two different human OSC lines (OSC1 and OSC2) that were established from the ovaries of two different subjects are shown as examples. Samples were resolved through agarose gels and stained with ethidium bromide to visualize DNA bands, with the resultant images inverted for photography and display purposes. (h,i) Immunofluorescence analysis of Prdm1 and PRDM1, Dppa3 and DPPA3, and Ifitm3 and IFITM3 expression (green) in mouse (h) and human (i) OSCs in MEF-free cultures. Cells were counterstained with DAPI (blue) and rhodamine-phalloidin (red) to visualize nuclear DNA and cytoplasmic F-actin, respectively. Scale bars, 50 μm.

  5. Spontaneous oocyte generation by cultured mouse and human OSCs.
    Figure 5: Spontaneous oocyte generation by cultured mouse and human OSCs.

    (ac) Oocytes formed by mouse OSCs in culture, as assessed by morphology (a), expression of the oocyte marker proteins Ddx4 and Kit (b; note the cytoplasmic localization of Ddx4 in the oocytes) and the presence of mRNAs encoding the oocyte marker genes Ddx4, Kit, Ybx2, Nobox, Lhx8, Gdf9, Zp1, Zp2 and Zp3 (c; samples were resolved through agarose gels and stained with ethidium bromide to visualize DNA bands, with the resultant images inverted for photography and display purposes). Scale bars, 25 μm. (d) Number of oocytes formed by mouse OSCs after passage and after seeding 2.5 × 104 cells in each culture well (values represent the number of oocytes generated over each 24-h block, not the cumulative number of oocytes; values are the mean ± s.e.m., n = 3 independent cultures, *P = 0.002 by analysis of variance (ANOVA) for the 48-h group compared to the 24-h and 72-h groups). (eg) In vitro oogenesis from human OSCs, with examples of the oocytes formed by human OSCs in culture (f, morphology; g, expression of the oocyte marker proteins DDX4, KIT, YBX2 and LHX8) and the number of oocytes formed after passage and after seeding 2.5 × 104 cells in each culture well (e; values are mean ± s.e.m., n = 3 independent cultures, *P = 0.002 by ANOVA for the 72-h group compared to the three other groups). The expression analyses of the oocyte marker genes (DDX4, KIT, YBX2, NOBOX, LHX8, GDF9, ZP1, ZP2 and ZP3) in human OSC-derived oocytes are shown in c, along with the results for mouse OSC-derived oocytes. Scale bars, 25 μm. (h) Immunofluorescence detection of the meiotic recombination markers DMC1 and SYCP3 (red; with a blue DAPI counterstain) in the nuclei of cultured human OSCs; human ovarian stromal cells were used as a negative control. Scale bars, 15 μm. (i) FACS-based ploidy analysis of cultured human OSCs 72 h after passage (for the mouse OSC analysis, see Supplementary Fig. 4).

  6. Human OSCs generate oocytes in human ovary tissue.
    Figure 6: Human OSCs generate oocytes in human ovary tissue.

    (ac) Direct (live-cell) GFP fluorescence analysis of human ovarian cortical tissue after dispersion, re-aggregation with GFP-hOSCs (a) and in vitro culture for 24–72 h (b,c) showing the formation of large single GFP-positive cells surrounded by smaller GFP-negative cells in compact structures resembling follicles (scale bars, 50 μm). (d,e) Immature follicles containing GFP-positive oocytes (brown, highlighted with black arrowheads, against a blue hematoxylin counterstain) in adult human ovarian cortical tissue injected with GFP-hOSCs and xenografted into NOD-SCID female mice (d, 1 week after transplant; e, 2 weeks after transplant). Examples of GFP-positive oocytes from multiple tissue replicates are shown. Scale bars, 25 μm. There are comparable follicles with GFP-negative oocytes in the same grafts. (f,g) All immature follicles in human ovarian cortical tissue before GFP-hOSC injection and xenografting (f) or that received vehicle injection (no GFP-hOSCs) before xenografting (g) (negative controls) contained GFP-negative oocytes after processing for GFP detection in parallel with the samples shown. Scale bars, 25 μm. (h) Dual immunofluorescence analysis of GFP expression (green) and either the diplotene-stage oocyte-specific marker YBX2 (red) or the oocyte transcription factor LHX8 (red) in xenografts receiving GFP-hOSC injections. Note that GFP was not detected in grafts before GFP-hOSC injection, whereas YBX2 and LHX8 were detected in all oocytes before and after injection. Sections were counterstained with DAPI (blue) for the visualization of nuclei. Scale bars, 25 μm.

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Author information

  1. These authors contributed equally to this work.

    • Yvonne A R White &
    • Dori C Woods

Affiliations

  1. Vincent Center for Reproductive Biology, Massachusetts General Hospital Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Yvonne A R White,
    • Dori C Woods &
    • Jonathan L Tilly
  2. Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Yvonne A R White,
    • Dori C Woods &
    • Jonathan L Tilly
  3. Department of Obstetrics and Gynecology, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

    • Yasushi Takai,
    • Osamu Ishihara &
    • Hiroyuki Seki

Contributions

Y.A.R.W., D.C.W. and J.L.T. designed the experiments, analyzed the data and wrote the manuscript. Y.A.R.W. and D.C.W. conducted the experiments. Y.T., O.I. and H.S. collected, cryopreserved and provided human ovarian cortical tissue. J.L.T. directed the project. All authors reviewed and approved the final manuscript for submission.

Competing financial interests

J.L.T. declares interest in intellectual property described in US Patent 7,955,846 and is a co-founder of OvaScience, Inc., and Y.A.R.W. and D.C.W. are scientific consultants for OvaScience, Inc.

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