Nanos2-null male gonocytes enter meiosis. (A–D) Embryonic testis samples prepared from Nanos2^+/− and Nanos2^−/− littermates at E14.5 and E15.5 were sectioned and immunostained with TRA98 (red) and anti-pH3 antibody (green). Arrowheads in D indicate male gonocytes in M phase. (E–H) Sections of testes from Nanos2^+/− and Nanos2^−/− littermates at E14.5 and at E15.5 immunostained with anti-Scp3 (green) and anti-Laminin 1 (red) antibodies. The inset in (H) shows a meiotic cell forming axial cores. (I–P) Sections from Nanos2^+/−Bax^+/−, Nanos2^+/− Bax^−/−, Nanos2^−/−Bax^+/−, and Nanos2^−/−Bax^−/− E17.0 testes immunostained with TRA98 (red) and anti-activated-caspase 3 (green) (I–L), or anti-Laminin 1 (red) and anti-Scp3 (green) (M–P) antibodies. Arrowheads in K indicate apoptotic male gonocytes. (Q,R) Chromosome spreads prepared from E17.0 Nanos2^−/−Bax^−/− testes and immunostained with an anti-Scp3 antibody. There were many cells at the zygotene (Q) but few cells at the pachytene (R) stage. (S–U) Sections from Nanos2^−/−Bax^−/− E17.0 testes immunostained with anti-Scp3 (green) and anti-Dmc1 (red) antibodies. DNA was labeled using the DAPI counterstain (blue). Bars: A (for A–D), E (for E–H), I (for I–L), M (for M–P), 100 μm; S, for S–U, 20 μm.

The forced expression of Nanos2 prevents meiosis and induces male characteristics in female embryonic germ cells. (A–E) Ovaries from control (A) and Nanos2-expressing (B–E) littermates at E16.5 were sectioned and immunostained with anti-Flag (green) and anti-Scp3 (red) antibodies. Arrowheads in B indicate Nanos2-positive female germ cells. (F–H) Sections were prepared from E13.5 ovaries of Nanos2-expressing mice and were immunostained with anti-Flag (green) and anti-Stra8 (red) antibodies. (I–Q) Sections were prepared from E16.5 ovaries of Nanos2-expressing mice and were immunostained with anti-Flag (green) (I,L,Q) and anti-dimethylated histone H3K9 (J), anti-TDRD1 (M), or anti-Dnmt3L (P) antibodies. Arrowheads in N indicate Nanos2-negative female germ cells. DNA was labeled by DAPI counterstaining (blue). Bars: A (for A,B), F (for F–H,O–Q,), 100 μm; C for C–E,I–N), 20 μm.

The relationship between Nanos2 and RA signaling. (A–F) Sections were prepared from E15.5 testes of Nanos2^+/− and Nanos2^−/− littermates, and were immunostained with TRA98 (red) and anti-Stra8 (green) antibodies. DNA was labeled with DAPI (blue). Bar: A (for A–F), 100 μm. (G) Real-time RT–PCR analyses of Stra8 in E12.5–E16.5 wild-type ovaries (green), and in E12.5–E14.5 testes of Cyp26b1^+/− (dark blue) and Cyp26b1^−/− (orange) littermates, and also in E13.5–E16.5 testes of Nanos2^+/− (blue) and Nanos2^−/− (red) littermates. (H,I) Real-time RT–PCR analyses of Cyp26b1 (H) in E13.5–E15.5 testes of Nanos2^+/− and Nanos2^−/− littermates, and of Nanos2 (I) in E12.5–E14.5 testes of Cyp26b1^+/− and Cyp26b1^−/− littermates.

Expression profiles of critical factors in the embryonic testes and a hypothetical model for the sexual differentiation of mouse germ cells. (A) Expression patterns for Cyp26b1 (blue), Stra8 (red), and Nanos2 (green) in E12.5–E15.5 wild-type, Cyp26b1-null, and Nanos2-null testes. In wild-type mice, Cyp26b1 is increased until E13.5 and gradually reduces thereafter (Fig. 2H; Bowles et al. 2006), whereas Nanos2 expression starts at around E13.5 and Stra8 is not induced. In Cyp26b1-null mice, Stra8 is up-regulated at E13.5 but is not induced until E14.5 in Nanos2-null mice, possibly due to the effects of Cyp26b1 (Fig. 2G). (B, left) RA signaling triggers meiotic initiation via the induction of Stra8 expression in female germ cells (pink). (Middle) In embryonic testes, Cyp26b1 inhibits RA signaling, which suppresses the entry of male gonocytes into meiosis and may provide the required conditions for the expression of Nanos2 until E13.5 (blue). (Right) After Cyp26b1 expression is decreased, Nanos2 inhibits meiotic initiation via suppression of Stra8 expression and induces male-type differentiation, which is indicated by the presence of the Dnmt3L and TDRD1 (blue).