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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 4. — Spermatogenesis is arrested at round spermatid stage in Smg6-cKO mice. (A) Quantification of different cell types in CTRL and Smg6-cKO seminiferous epithelium. Quantified cell types included SOX3-positive spermatogonia (Spg), γH2AX-stained sex body-positive spermatocytes (Spc), round spermatids (RS) and elongating spermatids (ES) that were recognized on the basis of nuclear morphology and their position in the epithelium, and SOX9-positive Sertoli cells (SC). Cells were counted from a minimum of 70 tubules across three biological replicates per genotype. Error bars represent mean ± SD, and p-value from Mann-Whitney U-test, *P < 0.05, ****P < 0.0001, NS = non-significant. (B) PFA-fixed testis sections were stained with Rhodamine-conjugated PNA (red) to visualize acrosome and DAPI (blue) to visualize chromatin. Scale bar: 20 μm. The lower panel shows electron microscopy of the acrosomal region of round spermatids in control and Smg6-cKO mice. Nu, nucleus; Cyt, cytoplasm. Stars indicate the acrosome, which is fragmented into three separate granules in cKO. Arrows indicate the borders of the acrosomal region. Scale bar: 1 μm. (C) The percentage of tubules containing at least one apoptotic spermatocyte or round spermatids in a whole testis cross-section. At least 90 tubule cross-sections from each of the 3 CTRL and 3 Smg6-cKO mice were analyzed. Two-sided Fisher's exact test was used to demonstrate a significant association between CTRL and Smg6-cKO mice with the percentage of tubules with apoptotic cells, ****P-value < 0.0001. (D) Quantification of apoptotic cells per seminiferous tubule cross-section in CTRL and Smg6-cKO testes. Error bars represent mean ± SD, and P-value from Mann–Whitney U-test, ****P < 0.0001. (E) Representative TUNEL staining images of apoptotic spermatocytes (upper panel, stage VII-VIII) and round spermatids (lower panel, stage X–XI) on Smg6-cKO testis sections, together with matched stages from control testes without apoptotic cells. Scale bar: 20 μm. (F) PFA-fixed epididymides of control and Smg6-cKO mice were stained with PNA (red) and DAPI (white). Selected round spermatids with positive acrosome staining indicated by white arrows. Scale bar: 20 μm. (G) Testis sections were immunostained with anti-ESPIN antibody (green) to visualize apical ectoplasmic specializations at stage VIII-IX of the seminiferous epithelial cycle. Scale bar: 50 μm. See also Supplementary Figure S4.