Loss of ar partially rescues nedd8?/? ovarian function. (A) Morphological comparison of ovaries and eggs from ar+/?nedd8+/+, ar?/?nedd8+/+, ar+/?nedd8?/? and ar?/?nedd8?/? female zebrafish at 4 mpf. Compared with the ovaries of the ar+/+nedd8?/? zebrafish (i.e. nedd8?/?; Fig. 1D), the ovaries of the ar+/?nedd8?/? zebrafish were filled with more oocytes in the midvitellogenic stage. (B) Histological analyses of the ovaries from female zebrafish with different genotypes at 4 mpf. Compared with oogenesis in the ar+/+nedd8?/? ovaries, oogenesis in ar+/?nedd8?/? ovaries was relatively normal. Green arrows indicate oocytes in different developmental stages. Bottom panels show magnification of the boxed areas in top panels. (C) Whole-mount images of embryos produced by different matings: ar+/+nedd8+/+ (?)×ar+/+nedd8+/+ (?); ar+/+nedd8?/? (?)×ar+/+nedd8+/+ (?); and ar+/?nedd8?/? (?)×ar+/+nedd8+/+ (?). The fertilization rate (number of fertilized eggs/total eggs laid×100) of the ar+/+nedd8?/? (?)×ar+/+nedd8+/+ (?) eggs was significantly lower than that of the ar+/?nedd8?/? (?)×ar+/+nedd8+/+ (?) eggs. Red arrows indicate degenerated embryos; green arrows indicate normal embryos. (D) Quantitation of the fertilization rates of different matings: ar+/+nedd8+/+ (?)×ar+/+nedd8+/+ (?) (n=10); ar+/+nedd8?/? (?)×ar+/+nedd8+/+ (?) (n=12); and ar+/+nedd8?/? (?)×ar+/+nedd8+/+ (?) (n=11). (E) Immunofluorescent staining of Vasa in PGCs of embryos (n=40) at 18?hpf from different matings: ar+/+nedd8+/+ (?)×ar+/+nedd8+/+ (?); ar+/+nedd8?/? (?)×ar+/+nedd8?/? (?); and ar+/?nedd8?/? (?)×ar+/+nedd8?/? (?). (F) The scatterplots represent PGC numbers in ar+/+nedd8+/+, ar+/+nedd8?/? and ar+/+nedd8?/?×ar+/? nedd8?/? embryos at 18?hpf. The numbers of PGCs were counted using a Leica dissection microscope based on the immunofluorescent density. EV, early vitellogenic stage; FG, full-grown stage; MV, midvitellogenic stage; PG, primary growth stage; PV, previtellogenic stage. Data are meanąs.e.m. **P<0.01, ****P<0.0001 (unpaired Student's t-test).
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