Fig. 7

Mutation of tp53 rescues the female-to-male sex-reversal phenotype of fancl mutants by reducing germ cell apoptosis.

(A) The distribution of individuals of different tp53 genotypes among fancl^-/- homozygous mutant progeny (n = 44) from an in-cross of double heterozygotes (fancl^+/-;tp53^+/-) is shown in a bar graph representing the number of females (purple bar) and males (green bars) distributed according to their tp53 genotypes (wild type, heterozygous or homozygous mutant). Rescue of female-to-male sex reversal was observed exclusively in fancl^-/- mutant homozygotes that were also homozygous for the tp53 mutation (n = 15): 11 fancl^-/-;tp53^-/- animals developed as females and 4 developed as males. No rescue was observed in fancl mutants that were either wild-type (n = 8; fancl^-/-;tp53^+/+) or heterozygous for the tp53 mutation (n = 21; fancl^-/-;tp53^+/-), which all developed as males. Total numbers of animals (n) are indicated on the graph per each sex in each genotype. (B,C) Hematoxylin and eosin staining of gonad sections of wild-type female (fancl^+/+;tp53^+/+, B) and rescued female doubly homozygous mutant (fancl^-/-;tp53^-/-, C) at adult stage, revealed the presence of morphologically normal ovaries in the rescued fancl^-/-;tp53^-/- females. Ovaries of both genotypes had oocytes at different stages of development (i.e.: IB, II, III, IV). Scale bar: 0.1 mm (B,C). (D,E,F) tp53 mutation reduces germ cell apoptosis in fancl mutants at 25 dpf. Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of fancl homozygous mutants simultaneously homozygous wild-type (D) or homozygous mutant for tp53 (E) at 25 dpf. Dashed lines outline gonad boundaries (D,E). (F) Bar graph representing the average number of Caspase-3-positive germ cells in fancl^-/-;tp53^+/+ (n = 5) and fancl^-/-;tp53^-/- (n = 5) at 25 dpf. Results showed that the average number of apoptotic germ cells was approximately three fold lower in doubly homozygous mutant animals (fancl^-/-;tp53^-/-; x- = 30±56) than their fancl^-/- mutant siblings that were wild-type for tp53 (fancl^-/-;tp53^+/+; x- = 105±71). This result shows that tp53 mutation decreased the number of apoptotic germ cells in fancl mutants at 25 dpf and demonstrates that the abnormal increase in germ cell apoptosis in fancl mutants that compromised the survival of developing oocytes was the mechanism responsible for the female-to-male sex reversal.