Phosphorylation state of a Tob/BTG protein, FOG-3, regulates initiation and maintenance of the Caenorhabditis elegans sperm fate program

Abstract

FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained for the continued production of sperm that is typical of the male sex. Vertebrate Tob proteins have antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to abrogate "antiproliferative" activity. Here we investigate FOG-3 phosphorylation and its effect on sperm fate specification. We found both phosphorylated and unphosphorylated forms of FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG-3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate specification was not only initiated but also maintained, resulting in continuous spermatogenesis in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program and that phosphorylated FOG-3 maintains that program for continued sperm production typical of males. We discuss implications of our results for Tob/BTG proteins in vertebrates.

Fig. 1.

FOG-3 is a phosphoprotein in vivo. (A) Genetic regulation of the C. elegans sperm/oocyte decision (simplified pathway). Boxed with a dashed black line are regulators of sex determination in all tissues; boxed with a solid black line are germline-specific regulators of sex determination, FOG-1 and FOG-3, which are terminal regulators of the sperm fate in both hermaphrodites and males. Blue proteins promote the sperm fate and red proteins promote the oocyte fate. (B and C) Schematics of adult germlines. Yellow, mitotically dividing germ cells, including germline stem cells; green, germ cells progressing through meiotic prophase I. (B) Adult male germlines make sperm (blue) continuously. (C) Adult hermaphrodite germlines contain a pool of mature sperm (blue), which were made during larval development, as well as developing oocytes (pale red). (D) Schematics of WT and alanine-substituted (4A) FOG-3 proteins. Yellow, Tob/BTG domain; green, predicted ERK docking site; black dots, consensus ERK phosphorylation sites with specific amino acid designated above; red asterisks, alanine substitutions at phosphorylation sites. (E) Phosphatase assay. FOG-3(WT)::3XFLAG was immunoprecipitated with anti-FLAG antibody from an extract prepared from fog-3; him-5; qEx[fog-3(WT)::3xflag] animals (mixture of XX and XO). The immunoprecipitated FOG-3(WT)::3XFLAG was treated with λ-phosphatase and phosphatase inhibitor, as noted, and analyzed by SDS/PAGE followed by immunoblotting. In α-FLAG panel, arrows mark phosphorylated (P-FOG-3) and unphosphorylated (FOG-3) forms of FOG-3. (F) FOG-3 phosphorylation state in vivo assayed by Western blotting. Worm extracts were prepared from fog-3; him-5; qIs[fog-3(WT)::1xflag] (lane 1) or fog-3; him-5; qEx[fog-3(4A)::1xflag] (lane 2) animals. In lane 2, phosphorylated FOG-3 is significantly reduced and the remaining FOG-3 migrates as if unphosphorylated.

Fig. 2.

FOG-3 is enriched in spermatogenic germlines. (A–D) Germlines dissected from animals harboring a fog-3(WT)::1xflag transgene and stained for FOG-3::1XFLAG (red) and DNA (blue). Dashed lines are mitotic zone/transition zone boundary or transition zone/pachytene boundary; red arrowheads mark the extent of elevated FOG-3::FLAG. (A) fog-3; qIs[fog-3(WT)::1xflag] XX hermaphrodite early L4. (B) fog-3; qIs[fog-3(WT)::1xflag] XX hermaphrodite late L4. (C) fog-3; fem-3(gf); qIs[fog-3(WT)::1xflag] XX hermaphrodite adult with a masculinized germline. (D) fog-3; qIs[fog-3(WT)::1xflag] XO male adult.

Fig. 3.

A fog-3 mutant rescue by FOG-3 wild-type and FOG-3 mutant transgenes. (A) Rescuing activity was scored in fog-3 null mutants harboring the transgene shown on left. Transgene schematics use the same conventions as detailed in Fig. 1D. Rescuing activity was scored in XX hermaphrodite adults (Left), XO male L4 larvae (Center), and XO male adults (Right). Hermaphrodite rescue indicates the percentage of self-fertile fog-3 XX animals, which made their own sperm (Left); larval male rescue indicates the percentage of fog-3 XO L4 larvae with any sperm (Center); adult male rescue indicates the percentage of fog-3 XO adults with continuous spermatogenesis (Right). Bar graphs on right depict the ratio of XO male rescue (continuous spermatogenesis in adults) to XX hermaphrodite rescue. See Table S1 for the rescue of each transgenic line. nd, not determined. (B and C) fog-3 mutant rescue by (B) fog-3(WT)::1xflag or (C) fog-3(T108A)::1xflag transgene. DAPI-stained adult male germlines (Top); schematics of the germlines (Middle); the average number of sperm produced at 1-d-old in the adult male germline (Bottom).

Fig. 4.

Model for control of the sperm fate specification. GSCs continuously produce progeny that differentiate as sperm or oocytes. Upper branch: The first set of GSC progeny adopts a sperm fate in both sexes. We propose that FOG-3 must be unphosphorylated at T108 to trigger this initial commitment. Lower branch: In males, GSC progeny continuously adopt the sperm fate in adults, whereas in hermaphrodites, germline sex switches to make oocytes in adults. We propose that FOG-3 must be phosphorylated at T108 and S260 in adult males to continue sperm fate specification and prevent the switch to oogenesis. FOG-3 could either be turned off as shown here (X), or left in its unphosphorylated state in adult hermaphrodites.