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Regeneration of Male Germline Stem Cells by Spermatogonial Dedifferentiation in Vivo

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Science  28 May 2004:
Vol. 304, Issue 5675, pp. 1331-1334
DOI: 10.1126/science.1097676
  • Fig. 1.

    Conditional loss of stat92E causes GSC differentiation. (A) The Drosophila testis apex. Ten GSCs (one shown) attach to the hub. Daughters (gonialblasts, GBs) are displaced from the hub, then undergo four mitotic (transit-amplifying) divisions to form 16-cell clusters of interconnected spermatogonia that exit mitosis and become spermatocytes. Fusomes (red) are spherical in GSCs and GBs; branched in spermatogonia and spermatocytes. Cyst cells (gray), produced by somatic stem cells (gray, at the hub) envelop GBs and form cysts. Color images are confocal sections through the testis apex. (B to H) Testes immunostained for germ cells (with α-Vasa, red), fusomes [with monoclonal antibody (mAb) 1B1, green], the hub (with α-FasIII, green, asterisk), and DNA [with 4′,6′-diamidino-2-phenylindole (DAPI), blue]. (B) Wild-type control shifted to 29°C for 2 days. GSCs contain spherical fusomes (small arrowhead). Spermatogonia and spermatocytes contain branching fusomes (large arrowhead). (C to H) stat92EF/stat92E06346 testes shifted to 29°C. (C) Day 0; a GSC-GB pair (outlined) has a round fusome (arrowhead). (D) Day 1; a four-cell spermatogonial cyst (three cells visible, outlined, arrow), two GSC-GB pairs (outlined, small arrowhead) contact the hub. (E) Day 2; spermatogonia enveloped by cyst cells (outlined, arrow marks one) surround the hub. Most contact it in at least one focal plane; the spermatocyte cyst (large arrowhead) does not. (F) Day 4; spermatogonial (arrow) and spermatocyte cysts (large arrowheads) surround the hub. (G) Day 6; a spermatocyte cyst (outlined, large arrowhead) and a few Vasa-negative cells (somatic stem cells or cyst cells, small arrowhead) remain. (H) Day 13; only the hub remains. (I and J) Testes immunostained to reveal the hub (α-Armadillo, red, asterisk), cell death (ApopTag, green), and DNA (DAPI, blue). (I) Wild-type and (J) stat92EF/stat92E06346 flies shifted to 29°C for 1 day contain dying spermatogonial cysts (outlined); cell death is not detected in GSCs. Scale bars, 10 μm.

  • Fig. 2.

    Differentiating spermatogonial cysts revert to stem cell identity when stat92E function is restored. (A to E) Confocal sections through the apex of testes stained to reveal the hub (α-Armadillo, red, asterisk), fusomes (mAb 1B1, red), four- or eight-cell spermatogonial cysts (α-BAM-C, green, outlined) and DNA (DAPI, blue). (A) Wild-type control. GSCs, GBs and two-cell cysts separate Bam-positive cysts from the hub. (B to E) stat92EF/stat92E06346 testes shifted to 29°C. (B) Day 1; Bam-negative and Bam-positive cells contact the hub. (C) Day 2, Bam-positive cysts surround hub. (D) Day 2, followed by 2 days at 18°C; Bam-negative cells reappear around the hub. (E) Day 2, followed by 10 days at 18°C; the testis apex appears normal. (F) The number of somatic cells (excluding hub cells) in serially reconstructed apical ends of stat92EF/stat92E06346 testes declines progressively at 29°C. (G) In stat92EF/stat92E06346 flies shifted to 29°C for 4 days, a normal number of GSCs returns after recovery at 18°C. (H to J) Confocal sections through the apex of testes as in Fig. 1. The rosette of GSCs (outlined), somatic stem cells (small arrowhead), spermatocytes (large arrowhead), and zone of spermatogonia (line) are indicated. (H) Wild type; GSCs contact the hub; followed by spermatogonia then spermatocytes. (I) stat92EF/stat92E06346 shifted to 29°C for 2 days, then 18°C for 2 days. Newly regenerated GSCs surround the hub; spermatogonia are absent. Spermatocytes (large arrowheads) fill the remainder of the testis apex. (J) After a 4-day recovery at 18°C, GSCs surround the hub; a zone of spermatogonia now separates them from spermatocytes (large arrowhead). Somatic stem cells are present between GSCs. Scale bars, 10 μm.

  • Fig. 3.

    Spermatogonial cysts break apart to form GSCs. (A to C) Confocal projection through testis apex, ring canals (α-Anillin, green) and fusomes (mAb 1B1, red) marked. (A) Testis from wild-type fly shifted to 29°C for 2 days. A GSC-GB pair (outline) completing cytokinesis shares a ring canal remnant (arrowhead) adjacent to the spectrosome. Four-, 8-, and 16-cell spermatogonial cysts (top to bottom) containing branching fusomes and multiple ring canals are outlined. (B) stat92EF/stat92E06346 shifted to 29°C for 2 days. Spermatogonial cysts (outlined) of 16 cells (top and middle) and 8 cells (bottom) contact the hub. (C) stat92EF/stat92E06346 shifted to 29°C for 2 days, then 18°C for 1 day. Ring canal remnants were found in four GSCs in this testis (one in this plane, small arrowhead, and inset). A mitotic 4-cell spermatogonial cyst (outlined, arrow) and a 16-cell cyst (outlined, large arrowhead) are visible. (D to F) Confocal projections through serially reconstructed testes; BrdU incorporation (green), fusomes (1B1, red), hub (FasIII, red, asterisk) and DNA (DAPI, blue) are shown. (D) Testis from stat92EF/stat92E06346 shifted to 29°C for 4 days, then labeled by BrdU incorporation in vivo. A 16-cell spermatogonial cyst with a branching fusome contacts the hub (outlined). (E) stat92EF/stat92E06346 shifted to 29°C for 4 days, then to 18°C for 2 days. Four GSCs with dot fusomes (small arrowhead) contact the hub (three are visible in this plane); these GSCs originated from an eight-cell cyst, producing a four-cell cyst in a lower focal plane (not visible). (F) Four labeled GSCs contact the hub (each containing a round fusome, arrowhead). An accompanying partial cyst (4 cells, outlined, arrow) and a 16-cell cyst (large arrowhead) are similarly labeled. Scale bars, 10 μm.

  • Table 1.

    Progression of GSC differentiation in stat92EF/stat92E06346 testes.

    Days at 29°C GSCs per testis Two- and four-cell cysts per testis Percentage testes with Testes scored
    GSCs Spermatogonia Spermatocytes
    0 10.00 ± 2.00 5.79 ± 2.30 100 100 100 25
    1 3.37 ± 0.93 11.26 ± 2.23 100 100 100 19
    2 0.25 ± 0.60 1.40 ± 1.48 22.50 100 100 40
    4 0.03 ± 0.24 0 1.40 50 50 73
    6 0 0 0 0 67 18
    13 0 0 0 0 0 17
  • Table 2.

    Labeled GSCs and accompanying partial spermatogonial cysts in stat92EF/stat92E06346 testes after BrdU incorporation and 2-day recovery at 18°C. Cysts were labeled to the same extent as adjacent GSCs and contained fewer than 16 cells.

    Days at 29°C Sample number Labeled GSCs Accompanying spermatogonia (n)
    4 1* 4 4
    2 3 13
    2 3 7 1
    4 5 4
    5 4 4
    6 4 5, 2
    7 4 4
    8 4 8, 4
    9 3 5
    10 2 6
    11 1 0
    12 1 0
    • * Fig. 3E.

    • 22% of testes contained one or two remaining GSCs but were distinguishable from GSCs derived from cyst breakdown, because the former contained a single labeled GSC and no accompanying labeled spermatogonia after recovery (samples 11, 12).

    • Fig. 3F.

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    Regeneration of Male Germline Stem Cells by Spermatogonial Dedifferentiation in Vivo
    Crista Brawley and Erika Matunis

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