Figure 6

3.5% 1,6 hexanediol exposure depletes spindle pole TACC3 in GFP CETN2-expressing spermatocytes resulting in meiotic spindle microtubule malformation, centriole abnormalities, and chromosome misalignment. Panel 1: TACC3 detection (A, E, I, M, Q) in GFP CETN2-expressing (B, F, J, N, R) meiotic spermatocytes (C, G, K, O, S; microtubules, red). Control untreated spermatocytes (A–C and E–G) show strong TACC3 spindle pole assembly (A, E: blue) marked by GFP-CETN2 centrioles (B, F: green, arrowheads) at assembling spindle microtubules (C, G: red, arrowheads). (D, H) Overlays of GFP CETN2 (green), MTs (red) and TACC3 (blue). Top insets: GFP CETN2 centrioles (green), microtubules (red), and DNA (blue). Bottom insets: details, GFP CETN2 centriole (green) and TACC3 (blue). (I–K) 3.5% 1,6-hexanediol spermatocyte exposure for 30 min removes TACC3 detection at spindle poles (I, M, Q), may reduce spindle microtubule density and organization (K, O, S: microtubules, red), disrupts GFP CETN2-expressing centrioles spindle pole localization (N: green) or detection (R: green), and impacts normal chromosome spindle alignment (left insets: L, P; lower inset, T: DNA, blue) (L, P, T): overlays of GFP CETN2 (green), microtubules (red) and TACC3 (blue). (L and P), middle and right insets: GFP CETN2 (green) and TACC3 (blue). Scale bars = 5 µm. Panel 2: TACC3 detection at the spindle poles of GFP CETN2-expressing spermatocytes exposed to 3.5% 1,6-hexanediol exposure for 30 min shows complete spindle pole removal of TACC3 (****; p < 0.0). Rescue from 1,6- hexanediol for 20 min before fixation showed a partial recovery not statistically significant from control cells (ns.; p < 0.235148) but significant from hexanediol exposure (****; p, 0.000004). Panel 3: measured fluorescent intensity of spindle pole microtubule reassembly is significantly greater in spermatocytes recovered from 3.5% 1,6-hexanediol exposure for 20 min that can restore TACC3 to the centrosomes compared to 10 µM MLN 8237 exposed spermatocytes recovered for 1-h that do not restore centrosomal TACC3 (****; p < 0.000034), although neither treatment approaches full spindle microtubule intensity of control spermatocytes (control vs hexanediol rescue: ****; p < 0.0005; control vs MLN 8237: ****; p < 0.000001).