Research ArticleCELL BIOLOGY

Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis

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Science Advances  12 May 2017:
Vol. 3, no. 5, e1602179
DOI: 10.1126/sciadv.1602179
  • Fig. 1 Generation of iPSCs from T. osimensis.

    (A) The female T. Osimensis found in the Amami-Oshima Island. Scale bar, 1 cm. (B) Sex is clearly distinguished by the distance between the anus and genitals. Scale bar, 1 cm. (C) Fibroblast cells were propagated from a tail tip in culture. Scale bar, 100 μm. (D) Phase-contrast images of primary colonies and tet-inducible hNANOG-Venus fluorescent images. A strong Venus fluorescent signal was observed in 5f1-1. Scale bar, 100 μm. (E) Dox-independent appearance of iPSC (4f1-1) colonies and their EOS-GFP fluorescence. Arrowheads indicate colonies observed after the withdrawal of Dox. Scale bar, 100 μm. (F) N2B27 3iL contained a B-Raf inhibitor, SB590885, which prevented iPSC (4f1-1) differentiation. Arrowheads indicate the differentiated cells that appeared in the N2B27 2iL (without SB590885) culture condition. Scale bar, 100 μm. (G) Karyotype analysis confirmed the normal chromosome number (2n = 25) in 4f1-1 and 5f1-1. (H) RT-PCR of endogenous expression of undifferentiated marker genes and exogenous genes in all iPSC lines established. (I) Teratoma with three germ layers was confirmed after transplantation of T. osimensis iPSCs (4f1-1 and 5f1-1) into SCID mice. Scale bar, 50 μm.

  • Fig. 2 Chimeric contribution of T. osimensis iPSCs in interspecific chimera.

    (A) A PB vector, pPB-CAG-Su9DsRed-ires-NeoR, was transfected into T. osimensis iPSCs (5f1-1). Scale bar, 100 μm. (B) Chimeric contribution of mouse ESCs (left, GFP) and T. osimensis iPSCs (right, Su9DsRed) in mouse embryos. Although mouse ESCs contributed to almost all of the bodies in 11.5-dpc embryos (top four embryos), T. osimensis iPSCs contributed partially in the embryonic bodies. Scale bars, 500 μm. (C) Photograph of newborn interspecific chimeras. Arrowheads indicate dark brown skin from the T. osimensis iPSC contribution. (D) Chimeric contribution of T. osimensis iPSCs in pups clearly visualized by DsRed signals. (E) Photograph of interspecific chimera at 3 weeks of age. (F) Photograph of an interspecific female chimera at 7 weeks of age. (G) Immunohistochemical detection of the chimeric contribution of T. osimensis iPSCs in several tissues. DsRed signals were sparsely distributed in several tissues. Scale bars, 200 μm. (H) Granular DsRed signal was detected in serial sections of an oocyte of a secondary follicle in a chimeric ovary (arrowhead). Left: Immunoreacted with normal immunoglobulin G (IgG) (negative control). Right: Immunoreacted with anti-DsRed antibody. Scale bar, 200 μm. The boxed regions are enlarged in the bottom images. Scale bar, 20 μm.

  • Fig. 3 Male germline contribution of T. osimensis iPSCs in an adult interspecific chimera.

    (A) Photograph of an interspecific male chimera at 7 weeks of age. Right panels indicate the testis from this chimeric male. Tubular contribution of T. osimensis iPSCs was not observed. (B) Testicular section of this interspecific chimera rarely detected T. osimensis iPSCs, which differentiated to putative germ cells as clustered signature (arrowheads). Dot-like signals, which indicate mitochondrial staining, are shown by anti-DsRed antibody (brown). The boxed region is enlarged at the right. Sections were counterstained with hematoxylin. (C) Immunofluorescence detection of DsRed and germ cell marker MVH. DsRed signals localized as a dot-like signature surrounding the cell nuclei (arrowheads) and indicate a mitochondrial localization. (D) Immunofluorescence detection of DsRed and a germ cell marker TRA98. DsRed signals localized at mitochondria. Almost all spermatocytes were stained by anti-TRA98 antibody at various staining intensities. (E) DsRed signal (arrowheads) detection in TRA98-negative elongating spermatids, which have sharply curved nuclei. (F) DsRed signal (arrowheads) detection of ACRBP/sp32 in acrosomes of round spermatids and (G) IZUMO1 in acrosomes of elongating spermatids. Merged images are shown on the right. The boxed region is enlarged at the far right. Sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI).

  • Table 1 Generation of interspecific chimeras using T. osimensis iPSCs.

    Chimera, DsRed+ and/or brown-haired; f, female; m, male; GC, germline contribution.

    Cell lineNumber of injected
    blastocysts
    Number of transplanted
    (recipients)
    Implanted or born
    12.5-dpc chimera/implanted (%)Chimera/pups (%)GC/chimera (adult)
    4f1-18670 (5)2/15 (13.3)4/26 (15.4)1 (f/m, 0:1)/4 (f/m, 2:2)
    5f1-1205185 (11)5/13 (38.4)9/25 (36.0)2 (f/m, 1:1)/9 (f/m, 4:5)
    Total291255 (16)7/28 (25.0)13/51 (25.5)3/13 (f/m, 6:7)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602179/DC1

    fig. S1. Determining appropriate culture conditions for T. osimensis iPSCs.

    fig. S2. LIF-dependent maintenance of naïve pluripotency of T. osimensis iPSCs.

    fig. S3. Chimeric contribution of T. osimensis iPSCs in interspecific embryos.

    table S1. Characterization of T. osimensis iPSC lines.

    table S2. PCR primers.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Determining appropriate culture conditions for T. osimensis iPSCs.
    • fig. S2. LIF-dependent maintenance of naïve pluripotency of T. osimensis iPSCs.
    • fig. S3. Chimeric contribution of T. osimensis iPSCs in interspecific embryos.
    • table S1. Characterization of T. osimensis iPSC lines.
    • table S2. PCR primers.

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