Figure 2.

Adjudin treatment induces defects in F-actin organization prior to germ cell release from the seminiferous epithelium that mimics spermiation. Adult rats (∼300 g b.w.) were treated with a single dose of adjudin at 50 mg/kg b.w. by oral gavage at 0 (controls; n = 5 rats). Thereafter, rats (n = 5) were terminated at specified time points at 6, 12, 24, and 96 h (i.e., day 4) for IF microscopy to visualize the organization of F-actin network across the seminiferous epithelium using FITC-conjugated phalloidin (green fluorescence; Invitrogen). Cell nuclei were visualized by DAPI (Invitrogen). A section of the images (boxed in red) on the second column was magnified and shown on the third and fourth columns; and a section of the images (boxed in yellow) on the fourth column was also magnified and shown on the fifth and sixth columns. The relative location of the base of the tunica propria was annotated by a dashed white line in the third column, and the relative location of the BTB was annotated by yellow arrowheads. The track-like structures (annotated by white arrowheads) conferred by F-actin were also shown in control testes. After adjudin treatment, F-actin that supported the BTB was no longer tightly aligned at the BTB (see white bracket in control testes on the third column), but diffusely localized in particular by 12 to 96 hours (see yellow brackets). These changes in F-actin distribution were semiquantified and shown in Supplemental Fig. 1 ^{(3.3MB, pdf)} . Also, the track-like structures conferred by F-actin were virtually nondetectable by 12 hours following adjudin treatment. Moreover, F-actin that appeared as bulb-like structures located at the concave side of spermatid heads was extensively mislocalized by 6 hours after adjudin treatment, moving to the concave side of spermatid heads and considerably diminished by 24 and 96 hours. This time-dependent F-actin disorganization at the apical ES induced by adjudin thus led to germ cell release from the epithelium, mimicking spermiation. Scale bars = 180, 70, and 30 µm in the micrograph in the first, third, and fifth column, which apply to corresponding images.