Differences in primordial germ cell number and shape diagnostic for the male and female gonad primordium anlage. A and B: Green fluorescence of the Olvas medaka primordial gonad at hatching stage (stage 39). Male (A), female (B). Both sexes have a significantly higher number of PGCs in the right PGC lateral clump. C: Comparison of the Olvas PGC phenotype versus genotype (Dmrt1bY) at hatching stage. D: Offspring obtained by mating a YAwr/YAwr female with an Olvas X/YHdrR male. E: Comparison of the Olvas primordial gonad phenotype at hatching stage versus genotype (YAwr or YHdrR). Identification of genetic sex in each individual was performed by PCR analysis for dmrt1bY presence or absence after genomic DNA extraction. The dmrt1bY fragments from either both Awr or Hd-rR-specific alleles were amplified with diagnostic primer sets. Scale bars: 7.5 μM.