Insect CPE synthases but not sphingomyelin synthases rescue spermatogenesis defects.

(A–C) Phase contrast microscopy of round spermatids where UAS hSMS1 (A), UAS Aedes CPES (B), and UAS Bombyx CPES (C) were ubiquitously expressed in cpes mutant background using tubulin Gal4. The scale bar is equivalent to 10 μm. (D) Measurement of cytokinetic defects in UAS-hSMS1, UAS-Aedes aegypti, and UAS-Bombyx mori CPES rescue testes; 4 independent experiments for UAS-hSMS1 (round spermatid count, n = 500), 2 independent experiments for UAS-Aedes aegypti (round spermatid count, n = 1,100), and 4 independent experiments for UAS-Bombyx CPES (round spermatid count, n = 1,900). (E–G) Confocal images of elongating spermatids immunostained with beta tubulin primary antibody and Alexa Fluor 568 secondary antibody (red) and DAPI for DNA (blue). UAS hSMS1 (E), UAS Aedes CPES (F), and UAS Bombyx CPES (G). (H–K) Sphingolipid analysis of lipids extracted from testis of wild type, cpes mutant, and various rescues expressed in germ cells using vasa-Gal4. In contrast to Figs 1 and 2, here we used whole male organ (testis, accessory glands, collecting ducts, and ejaculatory bulb) for lipid extraction. (H) The amounts of CPE and its subspecies in wild type, cpes mutant, and various rescues are shown. (I) Ceramide and its subspecies are shown in picomoles per 100 μg of carbon. (J) Hexosylceramide subspecies amounts are shown in bar diagram. (K) Amount of SM and its subspecies are shown as picomoles per 100 μg of carbon. Fluorescent blue green part of the bar represents SM species with net 2 double bonds in them. (L) TLC of lipids extracted from 50 pairs of testes from each genotype. The position of SM on TLC is indicated by an arrow. Original uncropped TLC image can be found in S1 Raw Images. DHSPH, SPH, SPD SFA, MUFA and PUFA. Statistical significance was calculated using mean, SD, and N in Prism 8. The 2-way ANOVA multiple comparison was used to calculate p-values where **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns p >0.05. Three independent biological replicates were taken for each sample and lipids were extracted from 250 male organs for each biological replicate. The data underlying the graphs shown in this figure can be found in S3 Data. DHSPH, dihydrosphingosine; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SD, standard deviation; SFA, saturated fatty acid; SM, sphingomyelin; SPD, sphingadiene; SPH, sphingosine; TLC, thin layer chromatography. (PDF)