Fig. 4

Loss of SSCs and/or parovaria in Hr39 mutant females does not inhibit the binding of SP to sperm but leads to retention of sperm and therefore elevated SP levels long term. A Western blot probed for SP. Lanes# 1: Fv, reproductive tract (RT) of 4 unmated females (negative control), 2: MAG, 1 pair of male accessory glands (positive control), 3: Ctl, sperm dissected from SR of 30 genetically matched control (BL64285) females mated to wild type (CS) males at 2 h ASM, 4: Exp, sperm dissected from SR of 30 mutant (BL64285) females mated to wild type (CS) males at 2 h ASM, 5: Ctl, sperm dissected from SR of 30 genetically matched control (BL64285) females mated to wild type (CS) males at 4 days ASM, 6: Exp, sperm dissected from SR of 30 mutant (BL64285) females mated to wild type (CS) males at 4 days ASM. Tubulin (Tub) served as loading control. B Graphical representation of the normalized levels of sperm-bound SP in Hr39 mutant (exp; pink bar) females relative to genetically matched control females (Ctl; blue bar) from the stock BL64285, at 2 h ASM (ns= nonsignificant; p=0.5328; df=2). C Graphical representation of the normalized levels of sperm-bound SP in Hr39 mutant (exp; pink bar) females relative to genetically matched control females (C; dark blue bar & dotted line) from the stock BL64285, at 4 days ASM (p**<0.01; df=2; AU stands for Arbitrary Units ). D Graphical representation of sperm counts in SRs of genetically matched control (C; blue bar) and Hr39 mutant (Exp; pink bar) females from BL64285 stocks mated to control ProtB-eGFP males (with eGFP-tagged sperm; p***=<0.001; n=15–20) and frozen at 8 days ASM. Error bars show Mean±SE. Sperm samples isolated from the seminal receptacle of E matched control females, F BL64285, Hr39 mutant females. The females were mated with CS males and frozen at 2 h ASM. G Corrected fluorescence (CF) intensity of SP bound to stored sperm in matched control (Ctl) and BL64285 females at 2 h ASM performed through immunofluorescence. Error bars show Mean±SE AU, F(13,13)=1.729, p=0.3360, ns= nonsignificant; n=14. Sperm samples isolated from the seminal receptacle of matched control females (H), BL64285, Hr39 mutant females (I). J Corrected fluorescence (CF) intensity of SP bound to stored sperm in matched control (Ctl) and BL64285 females at 4 days ASM performed through immunofluorescence. Error bars show Mean±SE AU, F(10,10)=60.63, p***<0.001; n=11. In all the immunofluorescence panels, sperm heads were stained with DAPI (blue) and anti-SP staining was visualized with Alexa fluor 488, staining the sperm tail (green) and sperm head (cyan; overlapping blue/green). The insets show the respective negative controls each panel. The bigger panels (H and I) in the 4day samples have transmitted light filter added to show the outline of sperm tail in the regions where SP was lowly detected (e.g., panel J); Bar = 20μm