The 9 dah-stage juvenile gonads were sexually undifferentiated
and consisted of large gonial germ cells and interspersed somatic
cells (Fig. S1). The progression of germ cell loss was histologically
examined. Thirty days after treatment, fish ovaries in the control
(27C), 32C, and 35C treated group had an ovarian cavity and
some oocytes at the perinucleolar stage indicating normal ovary
differentiation, whereas gonads of the 37C treated group consisted of few degenerating germ cells and supporting somatic cells (-figure not shown). Forty-five days after treatment, ovaries of the
control, 32C and 35C treated fish had ovarian cavities and
oocytes at the perinucleolar stage (Fig. 2A), whereas gonads of all
37C treated fish were devoid of germ cells (Fig. 2B and C). The
somatic cells did not show any morphological changes. To examine
the reversibility of sterility, 10 fish at each sampling time were
transferred to the tank with normal hatchery water temperature
(25–28C) and raised for additional six months. All fish in the control, 32C and 35C groups that were initially treated for 30 and
45 days had large, normal paired ovaries with mature oocytes after
extended rearing at control temperature (Fig. 2D and E). In the
37C, 30-day treatment group, two out of 10 fish had normal ovaries, six fish had abnormal ovaries with degenerated germ cells; and
two fish had thread-like paired gonads without germ cells (figure
not shown). In contrast, all fish in the 37C 45-day treatment
group had thread-like paired gonads with no germ cells (Fig. 2G
and H). All germ-cell-depleted ovaries had ovigerous lamellae
and follicular somatic cells. To confirm the absence of germ cells
in the gonads, we immunohistochemically examined ovary sections with the antibody against vasa. The oocytes in the control
ovaries had vasa positive immunoreactivity, whereas ovaries of
the 37C group fish did not contain germ cells and had no any
immunoreaction (Fig. 2F and I)