Fig. 3: piRNA loading is required for MIWI interaction with TDRD6 and transportation to the CB during male germ cell differentiation.

a Co-immunostaining of TDRKH (red) and TDRD6 (green) on testis sections from adult wildtype mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). PS pachytene spermatocytes, DS diplotene spermatocytes, RS round spermatids. Scale bar, 10 μm. b Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype and Tdrd6^−/− mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites. Scale bar, 10 μm. c, d Co-IP assay of the association of MIWI with TDRD6 and TDRKH in mouse testes from adult wildtype (c and d, lanes 3 and 4), Miwi^YY/YY (c, lanes 5 and 6) and Miwi^YK/YK (d, lanes 5 and 6) mice. Anti-MIWI IP pellets (c and d, lanes 4 and 6) were immunoblotted by the indicated antibodies, with testicular lysate (c and d, lanes 1 and 2) and IgG IP (c and d, lanes 3 and 5) serving as positive and negative controls, respectively. Quantification of blot intensity of indicated proteins in anti-MIWI IP pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. e Co-immunostaining of MIWI (red) and TDRD6 (green) on testis sections from adult wildtype, Miwi^YY/YY and Miwi^YK/YK mice using confocal microscopy, with nuclei counterstained by DAPI (greyscale). White arrowheads indicated colocalization sites. Scale bar, 10 μm. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.