Fig 5. CifA, CifB, and the protamine deficiency are transferred with the mature sperm to the female reproductive tract.

(A) Schematic representation of Drosophila melanogaster female reproductive system. Mature oocytes leave the OV and reach the UT, where they can be fertilized prior to being laid. Sperms from males are stored in specialized organs—SP and SR shown in the box, which open into the UT for fertilization to occur. Schematic is created with BioRender.com. (B) Transgenic cifAB-expressing and wMel− males were crossed to wMel− females. Four hours postfertilization, sperms isolated from females were decondensed and immunostained for localizing CifA (green) and CifB (red). DAPI stain (blue) was used to label nuclei. CifA is absent in sperm heads (empty arrowheads) and puctae are seen along the sperm tails (arrows). CifB is present in apical acrosomal tip of all of the sperm heads (solid arrowheads), with more distant signal in the more decondensed sperm nuclei. No Cifs are present in the sperms transferred from wMel− negative control males. (C) Individual sperm intensity quantification shows that protamine deficiency of sperms from wMel+ and transgenic cifAB males persists after transfer in the females compared to wMel− males. Sperm protamine deficiency from transgenic cifAB males also persists in the reproductive tract of wMel+ females. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant (p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p-values are reported in S1 Table, and raw data underlying this panel can be found in S1 Data file. (D) Representative images of CMA3-stained mature sperms (arrows) transferred from wMel−, wMel+ and transgenic cifAB males in wMel− and wMel+ female reproductive systems are shown. CMA3, chromomycin A3; OV, ovary; SP, spermathecae; SR, seminal receptacle; UT, uterus.