Figure 4

GFP CETN2-expressing spermatocytes exposed to MLN 8237 Aurora A kinase inhibitor significantly depleted spindle pole TACC3, inducing spindle microtubule abnormalities, reduced spindle lengths, and misaligned chromosomes but not γ-tubulin spindle pole intensity. Panel 1: GFP CETN2-expressing spermatocytes (green, arrowheads: A, B, D, E, G, H) in untreated controls (A–C), 10 µM MLN 8237 anti-Aurora A kinase inhibitor (D–F) or after rescue from 10 µM MLN 8237 exposure (1 h; G–I). MLN 8237 1 h exposure removed spindle pole TACC3 (blue; E, F), disrupted bipolar spindle organization (red; E, F), and chromosome alignment (D: blue) compared to untreated spermatocytes (A–C), but recovery experiments did not restore spindle pole TACC3 (H, I; blue) or chromosome alignment (G: blue) despite partial spindle kinetochore microtubule recovery (H, I: red, arrows). Insets: (B, E, H) GFP CETN2-expressing centrioles (green), microtubules (red) and spindle pole TACC3 (blue). Panel 2: MLN 8237 impact on spindle pole γ-tubulin. GFP CETN2-expressing spermatocytes (A, D, G: green, arrowheads) in untreated controls (A–C’), 10 µM MLN 8237 anti-Aurora A kinase inhibitor (1 h; D–F’) and after rescue from 10 µM MLN 8237 exposure (1 h; G–I’). MLN 8237 depleted spindle pole TACC3 (E, E’; blue) induced chromosome misalignment (D: DNA, blue), but not γ-tubulin spindle pole detection (F, F’: red) compared to control spermatocytes (A–C’). MLN 8237 recovery experiments did not restore spindle pole TACC3 (H, H’, I, I’: blue), DNA realignment (G: DNA, blue) or significantly impact γ-tubulin detection (G, I, I’: red). Panel 3: Analysis of spermatocyte MLN 8237 exposure showed the lowest concentrated tested (500 nM) significantly reduced spindle pole TACC3 compared to controls (****; p < 0.00009). Panel 4: γ-tubulin spindle pole intensity measured at the highest MLN 8237 concentration tested (10 μM; 1 h) did not impact γ-tubulin compared to controls (ns; p < 0.1485) but slightly reduced γ-tubulin intensity compared to controls after 1 h rescue (*; p < 0.0113), suggesting poor recovery from MLN 8237pleiotropic effects during meiosis. Panel 5: Spermatocyte spindle pole-to-pole lengths were significant from controls after 500 nM MLN 8237 exposure (*; p < 0.0152) or 1 h rescue experiments (*; p < 0.0158). All scale bars = 5 µm.