Effects of <i>cyp19a1a</i> and <i>cyp19a1b</i> Knockout on Germ Cell Kinetics During Gonadal Sex Differentiation in Medaka

In medaka (Oryzias latipes), the first morphological sex difference is germ cell number before hatching, which is determined by the dmy gene on the Y chromosome. This study aimed to clarify whether zygotically synthesized estrogen influences the kinetics of germ cell number during early gonadal sex differentiation. We established disruptive mutants of the estrogen-synthesizing enzyme aromatase by knocking out cyp19a1a (Δcyp19a1a) and cyp19a1b (Δcyp19a1b) as well as double disruptive mutants (Δcyp19a1s DKO) from each individual knockout using CRISPR/Cas9. Δcyp19a1s DKO XY and XX adult fish at 90 days post-hatching (dph) exhibited basal levels of estradiol-17β. At hatching (0 dph: stage 39), WT XX fry had significantly more germ cells than WT XY fry, and gonial cells were the most advanced germ cell stage across both sexes. Germ cell number and gonadal histology in Δcyp19a1s DKO mutants resembled those of WT fry. At 10 dph, germ cell number and gonadal histology were also similar between WT and Δcyp19a1s XY fry. In Δcyp19a1s DKO XX fry, diplotene oocytes and the total number of germ cells were significantly lower compared with WT. Exposure to 17α-ethynylestradiol rescued the reduction in diplotene oocytes in Δcyp19a1s DKO mutants to levels comparable to the control, resulting in the rescue of total germ cell number. Overall, our findings suggest that zygotically synthesized estrogen does not affect sex differences in germ cell number as the initial morphological sex difference but partly facilitates the differentiation from pachytene to diplotene oocytes.