Figure 2.

Immunodistribution of LC3 and p62 proteins in spermatozoa from normal and cryptorchid patients. (a) Faint anti-LC3 immunoreactivity (green) is detected in spermatozoa head and tail including axial filament (type 1 staining); in head equatorial segment (arrow), acrosome and the midpiece of the tail (type 2 staining). Note no staining in the axial filament and the end piece of flagellum in type 2 spermatozoa (arrowhead). (b) Pronounced anti-LC3 immunostaining is detected in acrosome and along the tail of cryptorchid spermatozoa including axial filament and end piece of flagellum (type 1 staining); very strong anti-LC3 immunostaining is present in the midpiece (b, arrowhead), but not in the axial filament in type 2 spermatozoa. (c) A control specimen treated with the primary antibody preincubated with LC3-blocking peptide (see Patients and Methods; upper panel). Bottom panel is the merge with DAPI-stained nuclei. (d) Histogram showing the relative quantity of type 1 and type 2 spermatozoa in the preparations from normal and cryptorchid patients. (e) Postacrosomal region (arrow) of control spermatozoa and the midpiece of the tail were stained with anti-p62 antibodies. (f) Anti-p62 immunostaining in cryptorchid spermatozoa. (g) Control specimen incubated with nonimmune rabbit serum instead of the primary antibody (upper panel). Bottom panel is the merge with DAPI-stained nuclei. (h) Histogram showing the relative quantity of spermatozoa stained with anti-p62 antibodies in the preparations from normal and cryptorchid patients. ^**P<0.05. LC3: microtubule-associated protein, 1A/1B-light chain 3; DAPI: 4,6- diaminido-2-phenylindole.