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Asz1 is essential for germ cell and gonad development in juvenile post-embryonic stages. A. Representative juvenile gonads from fish at 6 wpf of the indicated genotypes. Ruler grades are 1mm, showing much smaller, thread-like, gonads of asz1-/- compared to wt ovaries and testes. The distribution of gonad morphology from two representative clutches is plotted in B. n = number of gonads. Bars are mean ± SD. C. Representative confocal images of ovaries and testes of the indicated genotypes as in A, labeled with Ddx4 (green), mAb414 (red) and DAPI (blue). Insets show single and merge channels of magnifications of the yellow boxes in zoomed out images. Scale bars are 50 μm and 10 μm in zoomed out and inset magnification images, respectively. Wt ovaries (n = 16) and testes (n = 14) exhibited normal developing oocytes and early spermatocytes, as well as oogonia and spermatogonia as indicated by Ddx4, with normal perinuclear piRNA granules, as indicated by mAb414 signals (white arrowheads in wt ovary). D. Representative confocal images of ovaries and testes of the indicated genotypes as in A, labeled with cCaspase3 (green), mAb414 (red) and DAPI (blue). Insets show single and merge channels of magnifications of the yellow boxes in zoomed out images. Scale bars are 50 μm and 10 μm in zoomed out and inset magnification images, respectively. White arrowheads indicate cCaspas3-positive apoptotic cells. n = 10 ovaries and 10 testes (wt). E.asz1-/- gonads labeled as in C-D. Top: Ddx4 (green), mAb414 (red) and DAPI (blue). asz1-/- gonads (n = 13) had only few Ddx4-positive germ cells which exhibited abnormal mAb414 signal that appeared as coalesced granules (white arrowheads in asz1-/- gonads). Bottom: Acetylated tubulin (green), β-Catenin (red) and DAPI (blue), as in C-D. n = 6 gonads. F. The number of apoptotic cells per gonad is plotted and is non-statistically significant between genotypes. n = number gonads. Bars are mean ± SD.
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