Fig. 6

Phenotyping of CFAP161 protein in the non-capacitated (NC) and in vitro capacitated (IVC) spermatozoa with and without proteasomal inhibition, including vehicle control. The ICC detection of CFAP161 (red) in NC (a1) and IVC, non-inhibited (a2) spermatozoa, including non-immune serum control (a3). Spermatozoa were co-stained for acrosomal presence with peanut agglutinin (PNA, green), and nuclear stain DAPI (blue). All fluorescence channels are superimposed with the differential interference contrast (DIC) brightfield channel. Scale bars represent 20 µm. The IBFC of formaldehyde fixed and Triton X-100 permeabilized NC and IVC spermatozoa with or without proteasomal inhibition, including a vehicle and negative, normal rabbit serum control of the total sperm fluorescence intensity (b1) and the sperm flagellum fluorescence intensity (c1). Immunofluorescence images of NC (b2) and IVC spermatozoa (b3), including normal rabbit serum (b4) obtained by IBFC to confirm labeling. The IBFC was performed in 4 replicates with consistent results. Statistical evaluation of the total sperm fluorescence intensity (b5), and the sperm flagellum fluorescence intensity (c2) was performed by using ANOVA with Sidak’s post hoc test (α = 0.05).