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. 2023 Nov 11;12(22):2611. doi: 10.3390/cells12222611

Figure 7.

Figure 7

Rescue of the phenotypes of Nup62RNAi spermatocytes, including the failure of meiotic division, loss of anti-MPM2 immunostaining in the cytoplasm and nucleus, and nuclear accumulation of CycB by simultaneous depletion of rux encoding a CKI. (a–d) Phase-contrast micrographs of a single intact cyst composed of spermatids at the onion stage after the completion of meiosis II or at a slightly later stage. (a) Normal control spermatid cyst containing 64 cells. (b) An intact spermatid cyst composed of 16 spermatids derived from Nup62RNAi spermatocytes without meiotic divisions. The spermatids harbor under-condensed chromatin in the nuclei. (c) An intact spermatid cyst containing > 64 nuclei with a smaller size and >64 Nebenkerns generated from ruxRNAi spermatocytes. (d) An intact-like spermatid cyst containing >16 Nebenkerns and multiple smaller nuclei (arrow) together with larger-sized nuclei (arrowhead), which were derived from spermatocytes harboring Nup62RNAi and ruxRNAi, suggesting that meiotic divisions occurred in some spermatocytes of the cyst. Nuclei stained with DAPI are colored in red. Round or slightly extended black structures adjacent to nuclei correspond to Nebenkerns, mitochondrial derivatives. Scale bar: 10 μm. (e–i) Anti-MPM2 immunostaining (red in (e–i), white in (e’–i’)) of spermatocytes at the pre-meiotic S5 or an earlier stage to monitor Cdk1 activation. Sa-GFP (green in (e–i), white in (e”–i”)) is a marker to identify the stage of the growth phase. DNA staining (blue in (e–i), white in (e”’–i”’)); anti-CycB immunostaining (white in (e””–i””)). Scale bar: 10 μm. (e,f) Control spermatocytes at the pre-meiotic stage (e) and ProI (f). (g) Spermatocytes harboring testis-specific depletion of Nup62 (bam>Nup62RNAi) at the pre-meiotic stage. (h,i) Spermatocytes harboring testis-specific simultaneous depletion of Nup62 and rux (bam>Nup62RNAi, ruxRNAi) at the pre-meiotic stage (h) and ProI (i). Stages of spermatocytes were determined using the Sa-GFP marker. (j) Frequencies of spermatocytes harboring MPM2 epitopes in control (bam>+), Nup62RNAi (bam>Nup62RNAi, LacZ), ruxRNAi (bam>ruxRNAi), and Nup62RNAi and ruxRNAi (bam>Nup62RNAi, ruxRNAi) spermatocytes. ** p < 0.01, n.s.: not significant; one-way ANOVA followed by Bonferroni’s post hoc comparison. Error bars indicate SEMs.