Fig. 2. MAP is a sperm-associated protein. Equal samples of spawned sperm (A, Lanes 2-9) were treated with Triton X-100 (TX-100) at final concentrations varying from 0.01% to 2.00%, respectively. Control (Lane 1) and detergent-treated spermatozoa (Lanes 2-9) were studied by SDS-PAGE (A) followed by western blot with anti-MAP (B) or anti-esterase S (C) antibodies. As shown in A, the 20 kDa band intensity of the treated sperm remains unchanged at all TX-100 concentrations used. (D) Resulting supernatants of control (Lane 1s) and TX-100-treated spermatozoa (Lanes 2s, 3s and 9s) were concentrated and subjected to SDS-PAGE followed by western blot with anti-MAP antibodies. (E) Graphical presentation of the MAP densitometric analyses of both the gel (A) and blots (B,C), showing the percentage of the protein resistant to extraction by TX-100. Note that the three profiles (A—C) have the same MAP retention dynamics. (F) Immunofluorescence localization of MAP in the mid-piece region of spawned spermatozoa (white arrows) using anti-esterase S antibodies. (G) Control reaction: no MAP-positive signals were detected in the sperm cells treated with the antibodies pre-absorbed by MAP. Bars, 3 µm.