Fig. 7
Phenotyping of TTR in the non-capacitated (NC) and in vitro capacitated (IVC) spermatozoa, with and without proteasomal inhibition including vehicle control. The ICC detection of TTR (red) in NC (a1) and IVC, non-inhibited (a2) spermatozoa, including non-immune serum control (a3); the insets show TTR channel only. Spermatozoa were co-stained for acrosomal presence with peanut agglutinin (PNA, green), and a nuclear stain DAPI (blue). All fluorescence channels are superimposed with the 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 vehicle control and negative immunolabeling control, normal rabbit serum control (b1). 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 IBFC results (b5) was performed by using ANOVA with Sidak’s post hoc test (α = 0.05). The relative amounts of TTR median fluorescence intensity are related to the non-capacitated sperm control with an assigned value of 100%. The statistical significance P < 0.05 is denoted with small letters.
