Fig. 9.

Confirmation of CABYR and SPATC1 as targets for serine phosphorylation in crocodile spermatozoa. Proximity ligation assays (PLA) were employed to confirm that representative proteins, CABYR and SPATC1, were substrates for phosphorylation in noncapacitated (Non-cap) and capacitated crocodile spermatozoa. This assay results in the production of punctate red fluorescent signals when target antigens of interest, i.e., (A,) CABYR and phosphoserine residues or (B,) SPATC1 and phosphoserine residues reside within a maximum of 40 nm from each other. These experiments were replicated on independent samples from three different crocodiles, and representative PLA labeling patterns are shown. Arrowheads in (B,) indicate PLA labeling of the neck of the flagellum. C,, Negative controls included the labeling of spermatozoa with paired antibodies against phosphoserine and ZPBP1, a protein that was not identified as a substrate for serine phosphorylation, and the omission of one of the primary antibodies from the initial incubation (i.e., phosphoserine only). As anticipated, neither of these negative controls generated positive PLA labeling of the sperm flagellum. They did however, result in discrete, nonspecific PLA labeling in the anterior region of the sperm head (arrows). Scale bar, 5 μm.