Cortactin immunostaining was diffuse throughout the Sertoli cell cytoplasm when incubated without beads (Fig. 1A). When incubated with beads, the pattern of intracytoplasmic staining was less diffuse and displayed areas of variable density (Fig. 1B). Cortactin immunostaining was not apparent around the phagocytized beads (Fig. 1B). The primary antibody deletion staining control resulted in the absence of reaction product (Fig. 1A inset).

Cortactin immunostaining was distributed throughout the cytoplasm of Sertoli cells cultured without beads (Fig. 1C) and with beads (Fig. 1D) and in both was less diffuse than observed in the 15 min cultures. Also, in both treatment groups, reaction product appeared denser at the periphery of the cell (Fig. 1C,D) than observed in the 15 min cultures (Fig. 1A,B). Cortactin immunostaining was not apparent around the phagocytized beads (Fig. 1D).

In cultures without beads, cortactin immunostaining was observed throughout the cytoplasm of the Sertoli cells (Fig. 1E). Although not quantified, there appeared to be less cortactin than that observed at 15 min (Fig. 1A) and 2 hr (Fig. 1C) and less noticeable peripheralization than observed at the 2 hr time period. The addition of beads (Fig. 1F) did not appear to modify the pattern of cortactin immunostaining as compared to the culture without beads (Fig. 1E).

Some 15 min, 2 hr, and 24 hr cultures were exposed to cytochalasin D. In the 15 min culture, no noticeable difference in the amount of beads was observed. In the 2 hr culture (Fig. 2A), fewer beads were seen in the cultures exposed to cytochalasin D. The amount of beads in the 24 hr culture also appeared to be less than the amount of beads in the cultures not exposed to cytochalasin D (Fig. 2B), as in the 2 hr cultures. In the 24 hr culture, the cortactin reaction product was punctate (Fig. 3), whereas the culture not exposed to Cytochalasin D (Fig. 3 inset) was not, indicating its localization with the cytochalasin D-disrupted F-actin 19.

Western blot analysis (Fig. 4) confirmed the specificity of the primary antibody in control cells (3T3 lysate) and in cultured Sertoli cells.

Sertoli cells were isolated from 16-day-old Sprague-Dawley rats (Harlan) by sequential enzymatic digestion with trypsin and collagenase, as previously described by Cameron et al 23. Briefly, testes were excised from prepubertal male rats, and the parenchyma was digested with routine sequential enzyme treatments (0.25% trypsin, followed by 0.20% collagenase). Cells were plated (<1.5 × 10⁶ cells/cm²) in 4 well chamber slides (Lab-Tek^®) precoated with Matrigel^® (1:5 dilution with supplemented medium). Sertoli cell viability at plating was >95%. Plated cells were incubated in DMEM:F12 medium supplemented with 50 ng/ml retinol (Acros) and 0.01 cc/ml insulin-transferrin-selenium (ITS; Sigma) at 39°C, in a humidified incubator with 5% CO₂-95% air, for 2 days, to expedite the removal of contaminating germ cells. The Sertoli cell cultures were then treated with a hypotonic solution of sterile 20 mM Tris-HCl buffer for 2.5 min at 37°C to remove any remaining germ cells, after which the pretreated cells were then placed in a humidified chamber at 33°C with 5% CO₂-95% air.

Pretreated Sertoli cell monocultures were incubated for 24 hours, after treatment with Tris-HCl, in supplemented DMEM:F12 medium and incubated with or without 0.1 μg/ml FSH (NIDDK-oFSH-19-SIAFP, 94× NIH-FSH-S1/mg; gift from NIDDK-NIH) in a humidified chamber at 33°C with 5% CO₂-95% air for an additional 24 hours.

At time 0 (4 days in culture), some pretreated Sertoli cell monocultures received 2 μM cytochalasin D (Sigma) 24, 0.8 μm latex beads (Sigma), or both 2 μM cytochalasin D and 0.8 μm latex beads. These pretreated Sertoli cell monocultures were incubated for 15 min, 2 hr, or 24 hr in supplemented DMEM:F12 medium in a humidified chamber at 33°C with 5% CO₂-95% air. No attempt was made to quantify bead uptake by Sertoli cells, although all cultures were observed by differential interference contrast microscopy after fixation and repeated washings to determine if the beads were in or on the cells. Control pretreated Sertoli cell monocultures received no cytochalasin D or latex beads.

Sertoli cells were fixed with 100% ethanol and immunostained for cortactin, as described by Wine and Chapin 25, using 10 μg/ml anti-cortactin (p80/p85), clone 4F11 (Upstate) as the primary antibody. Two secondary antibodies were used. The first one was rat antimouse IgG1 heavy chain:biotin (1:200; Serotec), which was conjugated to streptavidin horseradish peroxidase (Zymed). Positive immunostaining was visualized with bright light by reduced DAB (Vector Labs). The second one was rat antimouse IgG1 heavy chain:FITC (1:200; Serotec). Positive immunostaining was visualized by ultraviolet light. Latex beads fluoresced when excited with 540 nm wavelength light, there was no counterstaining, and appropriate positive (3T3 cells) and negative (primary antibody deletion) staining controls were used.

SDS-PAGE gel electrophoresis was performed to verify the antibody specificity. 20 μg protein was loaded onto the gel. Cold cell lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; Complete™ Mini protease inhibitor cocktail (Roche); 1 mM Na₃VO₄; 1 MM NaF) was added to the cell cultures, and the cells were detached using a disposable cell scraper. The cell suspension was lysed on an orbital shaker for 15 min at 4°C, after which the lysate was centrifuged at 14,000 × g for 15 min at 4°C. The supernatant was collected for Western blot analysis.

Lysates were separated on a 7.5% Tris-HCl Ready Gel (BioRad) and transferred to nitrocellulose using a semi-dry blotting apparatus. The membrane was immunostained with 1 μg/ml anti-cortactin (p80/p85), clone 4F11 (Upstate), as the primary antibody, and anti-mouse IgG (H&L) AP conjugate (Promega), as the secondary antibody. The target protein on the membrane was visualized by Western Blue^® Stabilized Substrate for Alkaline Phosphatase (Promega).