Figure 4.

Testes from Duba-null flies display morphological defects and reduced caspase activity. (a) Testes lysates from 1- to 2-day-old homozygous Duba¹⁰⁷flies or controls were immunoblotted to confirm a complete lack of DUBA protein. * indicates a nonspecific signal of the α-DUBA antiserum. Actin levels are shown as loading control. (b and c) Seminal vesicles of Duba¹⁰⁷ testes do not contain mature sperm. Seminal vesicles of control (b) and Duba¹⁰⁷ (c) testes, stained with DAPI, are shown. White arrows indicate round-shaped nuclei from cells of the sheath of the seminal vesicles; red arrow shows single needle-shaped nuclei of individualised spermatids that are only present in control testes. Scale bar: 50 μm. Repeated three times. (d and e) Confocal projections of testes of 1-day-old control (d) and Duba¹⁰⁷ (e) flies reveal structural differences in individualising and elongated spermatids. CB, cystic bulge; IC, individualisation complex; WB, waste bag. Testes were stained with DAPI (blue), phalloidin (magenta) and antibodies against active caspase (cDcp1, green) and polyglycylated tubulin (AXO49, red). Enlarged in (d'–d''''): individualisation complex and cystic bulge. Enlarged in (e'–e''''): Elongated spermatids of Duba¹⁰⁷ testes lack IC and CB and show degrading cysts (e'''', white arrowhead). Scale bar: 100 μm in (d and e), 50 μm in (d'–d'''') and (e'–e''''). Repeated four times, four to six testes per condition each time. (f) Caspase activity in lysates from Duba¹⁰⁷ testes was measured by DEVDase assay in vitro and compared with control lysate or lysate treated with the caspase inhibitor z-VAD-fmk. Means of three independent experiments and S.E.M. are shown. RFU, relative fluorescence units