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. 2022 Oct 19;50(20):11470–11491. doi: 10.1093/nar/gkac900

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — SMG6 localizes to the CB in round spermatids. (A) Western blotting of different tissues using anti-SMG6 antibody with anti-GAPDH as loading control. T, testis; E, epididymis; O, ovary; U, uterus; K, kidney; S, spleen; Lu, lung; Li, liver. (B) Relative expression of SMG6 during the first wave of spermatogenesis. Testis samples were collected from juvenile mice at different time points (1, 2, 3, 4 and 5 weeks) and adult mice (7 and 10 weeks). Two independent sample sets were included. Anti-SMG6 western blots were quantified with ImageJ software using anti-GAPDH for normalization. Error bars represent mean ± standard deviation (SD). (C) Representative western blot image of (B). (D) Immunohistochemistry of testis sections from 1, 2, 3, 4 and 5 weeks (w) old mice using anti-SMG6 antibody or no primary antibody (CTRL). Arrows point at selected examples of cytoplasmic SMG6 signal in round spermatids. Asterisks: elongating spermatid bundles. Scale bar: 5 μm. (E) IF of testis sections using anti-SMG6 antibody (red). PSpc, pachytene spermatocyte; RS, round spermatid; ES, elongating spermatid; M, meiotic metaphase plate; 2nd Spc, secondary spermatocyte. Arrows point at selected cytoplasmic granules. Scale bar: 10 μm. (F) Co-IF of SMG6 (magenta) and DDX4 (green) in round spermatids (upper panel, white arrow) and in late pachytene spermatocytes (lower panel, dashed circle). Scale bar: 10 μm. (G) Co-localization of SMG6 (magenta) with DDX4 (green) in human round spermatids (upper panel) and spermatocytes (lower panel). Arrows point at selected CBs. Scale bar: 10 μm. (H) SMG6 co-localizes with DDX25 and PIWIL2 to CB precursors in late pachytene spermatocytes (stage XI). SMG6 in magenta, DDX25 (left panel) and PIWIL2 (right panel) in green. Confocal 4-channel imaging was used to detect all three specific antibodies and DAPI in the same slides and artificial colors to each channel rendered afterwards using Adobe Photoshop. Scale bar: 10 μm. (I) The area indicated by a white box in panel H with higher magnification. Upper panel: co-localization of SMG6 with CB-specific DDX25. Lower panel: partial co-localization of SMG6 with PIWIL2. Co-localization points are marked by arrows. White dashed lines indicate examples of PIWIL2-positive (but DDX25- and SMG6- negative) IMCs. See also Supplementary Figure S1.